BIO 241
CELL BIOLOGY LAB MANUAL
INSTRUCTOR: Nesrin Özören
LAB ASSISTANTS: Sibel Uğur
Tolga Aslan
Tuncay Şeker
Çiğdem Atay
Boğaziçi University
Department of Molecular Biology and Genetics
2007-2008 FALL
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BOĞAZIÇI UNIVERSITY
DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS
2007-2008 FALL
TABLE OF CONTENTS
Grading of the Course..................................................................................................................... 2
A PREVIEW: THE WORLD OF THE CELL GUIDELINES .................................................... 3
How to Keep a Lab Notebook ........................................................................................................ 4
How to Write a Lab Report .............................................................................................................. 5
Experiment 1
: Microscopic Measurements.............................................................................. 10
Experiment 2
: Plasma Membrane ............................................................................................ 14
Experiment 3
: Cellular Fractionation ....................................................................................... 17
Experiment 4
: Analysis of Subcellular Fractions I .................................................................. 21
Experiment 5
: Analysis of Subcellular Fractions II ................................................................. 26
Experiment 6
: DNA Extraction From Bovine Spleen.............................................................. 29
Experiment 7
: Mitosis and Cytokinesis ................................................................................... 32
Experiment 8
: Cell Culture ...................................................................................................... 35
Experiment 9
: Analysis of Polysaccharides ............................................................................. 39
Experiment 10 : Cellular Carbohydrates ..................................................................................... 43
TT 7-8
Experiment 1
Experiment 2
Experiment 3
Experiment 4
Experiment 5
Experiment 6
Experiment 7
Experiment 8
Experiment 9
Experiment 10
Microscopic Measurements
Plasma Membrane
Cellular Fractionation
Analysis of Subcellular Fractions I
Analysis of Subcellular Fractions II
DNA Extraction From Bovine Spleen
Mitosis and Cytokinesis
Cell Culture
Analysis of Polysaccharides
Cellular Carbohydrates
Review
Lab Final
ThTh
2-Oct-07
4-Oct-07
9-Oct-07
11-Oct-07
16-Oct-07
18-Oct-07
23-Oct-07
25-Oct-07
30-Oct-07
1-Nov-07
6-Nov-07
8-Nov-07
13-Nov-07
15-Nov-07
20-Nov-07
22-Nov-07
27-Nov-07
29-Nov-07
4-Dec-07
6-Dec-07
11-Dec-07
13-Dec-07
With course final
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GRADING OF THE COURSE
Your lab grade makes up 20% of your course grade and it should be at least 45/100 in
order to be successful in Bio241.
Lab Reports
10/20
(submission at the beginning of following lab session. –1 for each day late and not
accepted after 3 days)
Introduction
1.5/10
Purpose
1.0/10
Materials
0.5/10
Procedure
1.0/10
Results-Discussion-Questions
5.0/10
References
1.0/10
(“Lab manual” as a reference is not accepted)
Quiz
3/20
Lab Final
7/20
Questions: please contact bio241lab@yahoo.com.tr
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THE WORLD OF THE CELL: A PREVIEW
The cell is the basic unit of life. Every organism either consists of cells or is itself a
single cell. Only as we understand the structure and function of cells can we appreciate both
the capabilities and limitations of living organisms, whether animal, plant or microorganism.
We are in the midst of a revolution in biology that has brought with it tremendous
advances in our understanding of how cells carry out the intricate functions necessary for life.
Particularly significant is the dynamic nature of a cell, as evidenced by its capacity to grow,
reproduce and become specialized and by its ability to respond to stimuli and to adapt to
changes in its environment.
Cell biology itself is changing, as scientists from a variety of related disciplines focus
their efforts on the common objective of understanding how cells work. The convergence of
cytology, molecular genetics and biochemistry has made modern cell biology one of the most
exciting and dynamic disciplines in contemporary biology.
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How to Keep a Lab Notebook
Guide for Students and Laboratory Instructors
Department of Molecular Biology and Genetics
The most important aspect of scientific investigation is keeping track of your work. There
is no point in performing work that will certainly be lost, as time efficiently erases memory,
people, and even single copies of anything. In a world of increasing standards, GLP (good
laboratory practice) is in itself becoming an area of expertise. A good lab notebook should
contain sufficient detail for any other person to be able to repeat the same experiment and
achieve the same results, reproducibility being a primordial requisite for a phenomenon to
become a scientific fact.
Legally, all proprietary rights conflicts are resolved primarily by investigation of lab
notebooks. In the case of industrial labs, notebooks belong to the laboratory and must include
details such as make and lot number of reagents, date of preparation of solutions, names of
persons that prepared them. Each page of the notebook is numbered, dated, signed by
investigator and countersigned by controller. In student labs, we need not go that far; the
minimum requirements are given below.
LABORATORY NOTEBOOK: Use a single, bound-page notebook, never use loose notepaper.
Always include experiment date and title. Make notes as you go along. Note everything you
use and do, keeping in mind the rule that someone else should be able to repeat what you did
by reading your notes. You may refer to published protocols/methods, but must note any
modifications or specific conditions. Note details of calculations, such as for solutions or
dilution series, including volumes actually used. Paste all figures, photos, printouts etc. in
notebook. Every observation is important.
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How to Write a Lab Report
Preparation and Evaluation Guide for Students and Laboratory Instructors
Department of Molecular Biology and Genetics
This guide provides a standard format in which all lab reports, in all courses, must be
written. The goal of the lab report, like a scientific paper, is to convey to your audience why
the experiment was done (background) what was done (materials and methods), what was
the outcome (results), the significance of the results (discussion), and the published sources
that assisted your experimentation or interpretation (references). Some courses may require
slight alterations or changes to this format, and these will be explained at the beginning of the
lab.
The basis for writing a good lab report, like writing a research paper, is an organized
record of the experiments and your results. A lab notebook is the place where experiments are
described in great detail, and the raw data is recorded.
LAB REPORT REQUIREMENTS
1. Keeping a detailed lab notebook is a prerequisite. Please see the attached Lab Notebook
Guidelines.
2. Reports must be original work. Although experiments may be carried out as a group, each
report must be written individually. Plagiarism will result in a grade of zero for the
report.
3. Reports must be type written and subjected to a spell check. Grammar and spelling
mistakes are the first indication of sloppy work.
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Lab Report Format
Each section must be included in each report, unless told differently by the lab
instructor.
I. Title page
A. Experiment name
B. Experimenter (author) and partners (co-authors)
C. Experiment date(s)
D. Evaluator
E. Submission date
II. Introduction
A. Provides a brief summary of the background and theory pertaining to the experiment
done.
B. Material for the introduction can be found in books, articles, your lab manual or the
internet (but only from reliable sites– be careful!). Any information that is not general
knowledge must be referenced appropriately. Plagiarism is easily recognized.
C. Must answer the questions of;
What was known before the experiment was done?
Why was the experiment carried out?
Was a hypothesis being tested? If so, the hypothesis must be specifically stated.
III. Purpose
Few sentences in order to answer;
“why was this experiment performed?”
IV. Materials and Methods
A. What materials and reagents were used?
B. What was done – step by step in your own words (not copied directly from lab
manual)? Diagrams and flow charts are welcome.
C. Alterations, mistakes or corrections must be included. Very important!!!
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V. Results
A. Informing your audience of the purpose of the experiment
“In order to measure the pH of X…” or “To ask whether the sample contained
enzyme X…”
B. State the direct outcome of the experiment or procedure.
note the difference between “what you see, what you think you see, and what you
think it means” (Moriarty, 1997)
C. Each experiment should answer a simple question
i.e. “what do the cells of an algae look like?” or “what is the concentration of cells
in an unknown sample?”). Each answer should be represented by the results of your
experiment both in table or figure AND in words. When writing a description, your
audience should be able to use your words to reconstruct what you observed.
D. The following questions, as suggested by Moriarty (pgs. 89-90) (Moriarty, 1997),
should be answered in the result section of any piece of scientific writing
Why did you conduct the experiment?
What did you do?
What did you see?
What does it mean? This point is stated in more detail in the discussion, but it can
be simply stated (one sentence) in the results.
E. Figures and tables must be included that show the data generated by your experiment.
Hand drawings and hand written calculations are acceptable, but they must be neat and
labeled.
Tables and figures must be numbered and titled.
Table titles appear at the top, figure titles at the bottom.
Tables: rows and columns are labeled.
Figures: axes are labeled and contain units.
VI. Discussion
A. Restate in the first sentence or two the purpose and findings of the experiment.
B. This section is the explanation of the results section.
C. Include explanations of unpredicted or inconsistent results.
D. Place the results into a setting.
E. Compare and contrast results with existing knowledge.
F. Explain why you think the results mean.
G. Referencing other studies is appropriate.
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H. Discussion should give the audience a general conclusion about the results and answers
to the questions posed in the introduction.
I. May refer to future experiments that can answer questions raised by this study. This is
not always appropriate.
What would you do next if you were to continue with this study?
What would you change if you were to do the experiment again?
VII.
References
A. ALL thoughts, data or ideas that are not your own must be referenced.
B. If something is a generally known fact, it does not need to be referenced. This includes,
but is not limited to,
Chemical molecular weights
Species names
Molecular composition of known compounds
C. Be very careful when using websites. Information from the internet can be misleading
or wrong, so you must be critical. Personal websites are not valid references. Any
website used will be highly scrutinized, and if untrustworthy or even questionable, this
will be detracted from your grade.
D. The reference must be given in the text with the name of the author and the year of
publication, and the full reference must be provided in the references section.
The following selection taken from a recent article in EMBO reports (Richard and
Pâques, 2000) demonstrates acceptable methods of referencing others work:
“In human diseases, it is common to observe expansions of more than twice the
original size. Such large expansions in yeast and humans could occur by successive
rounds of unwinding/re-invasion of the donor sequence by the newly synthesized
strand, allowing DNA synthesis to proceed more than once within the repeats
(Pâques et al., 1998; Figure 2c). An interesting case of large contraction of a CAG
repeat was described during transmission of a myotonic dystrophy allele. O'Hoy et
al. (1993) reported a large reduction of the number of CAG triplets associated with
what they called a ‘discontinuous gene conversion event’. The resulting allele was a
patchwork of both maternal and paternal alleles. Buard and Vergnaud (1994),
Debrauwère et al. (1999) and Tamaki et al. (1999) also found complex
recombination events in minisatellites.”
E. References must follow standard format. Examples given below.
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Journal: Author, (year) title. Journal volume(issue), pages.
ex.: Morehouse, S.I., Tung, R.S., Rodriguez, J.-C., Whiting, J.R. and Jones, V.R.
(1993) Statistical evidence for early extinction of reptiles due to the K/T event.
Journal of Paleontology 17(4), 198-209.
Book: Author (year) title, number of pages. Edition number. Edition series, editor.
Issue. Number of volumes. Publisher, city.
ex.: Billoski, T.V. (1992) Introduction to Paleontology, 212 pp. 2nd ed. Trans. A.
Translator. Series on Paleontology, edited by B.T. Jones, 6. 12 vols. Institutional
Press, New York.
Book with referred Chapter: Author (date) title. In: book editors (Eds), book title,
edition pages. Volume. Number of volumes. Publisher, City.
ex.: Grosjean, F.O. and Schneider, G.A. (1990) Greenhouse hypothesis: Effect on
dinosaur extinction. Trans. M.A. Caterino. In: N.R. Smith and E.D. Perrault (Eds),
Extinction, 3rd ed., pp. 175-189. Vol. 2. 5 vols. Barnes and Ellis, New York.
Website: Author (date) Title of page or article (web site).
ex.: Gutkind, J. S. (2000). Regulation of mitogen-activated protein kinase signaling
networks by G protein-coupled receptors (http://www.stke.org).
VIII. Appendices
A. Calculations
B. Detailed information about equipment used if required.
C. Answers to questions posed in the lab manual.
References
Moriarty, M.F. 1997. Writing science through critical thinking. Jones and Bartlett Publishers, Inc.,
London.
Richard, G.-F., and F. Pâques. 2000. Mini- and microsatellite expansions: the recombination
connection. EMBO Reports. 1:122-126.
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EXPERIMENT NUMBER: 1
“MICROSCOPIC MEASUREMENTS”
1.1. INTRODUCTION
The light microscope (Figure 1.1) employs visible light to detect small objects and it is
probably the most well-known and well-used research tool in biology.
Figure 1.1. Diagram of a typical light microscope, showing the parts and the light path
Types of Light Microscope
The bright field microscope will be used in this course.
Visible light is focused through a specimen by a condenser lens,
and then is passed through two more lenses (objective and ocular
lenses) placed at both ends of a light-tight tube (Figure 1.2). The
latter two lenses each magnify the image. A third lens system
located in the front part of the eye generates a real image on the
retina. Limitations to what can be seen in bright field microscopy
are related to resolution, illumination, and contrast. Resolution can
be improved using oil immersion lenses, and lighting and contrast
can be dramatically improved using modifications such as dark field, phase contrast, and
differential interference contrast.
Magnification, the degree of enlargement of the image of the object compared to its
real size. It is provided by a two lens system; ocular lens (8 or 10X) and objective lens
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(4, 10, 40 or 100X). Total magnification is the product of these two magnifying power
values.
Resolution, a measure of the clarity of an image.
Resolving power is the ability of an optical instrument to show two objects as
separate. For example, what looks to your unaided eye like a single star in the sky may
be resolved as two stars with the help of a telescope. Any optical device is limited by
its resolving power.
Contrast, is the ability to determine same particular detail of specimen against its
background. In a bright field light microscope, adjustable condenser with aperture
diaphragm control or adding dyes may increase contrast of a transparent specimen.
The maximum magnification obtained through a light microscope is 400X, in other
words the closest two distinct points can be and still be resolved is 0.2 micrometer (µm) about
the size of the smallest bacterium (1 µm = 10-6 m). This limitation is the result of light being
diffracted by the object under observation and because diffracted light interferes with the
image.
Microscopic Measurements
The linear size of a specimen observed in the microscope is best expressed in µm and
not as a total magnification. The actual size of an object viewed under the microscope can be
estimated, based on the fact that “the field of view for a given microscope and a given
combination of lenses will have a constant diameter”. A measuring device “hemocytometer”
have been provided for you to measure the field of view.
0.05 mm
0.05 mm
Figure 1.3.
Hemocytometer under
light microscope
Focus on the SMALLEST
SQUARES in the middle
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A device used for cell counting is called a counting chamber. The most widely used
type of chamber is called a hemocytometer (Figure 1.3), since it was originally designed for
performing blood cell counts. However, in this experiment hemocytometer is provided in
order to measure the field of view.
You can compute the size of the field for the higher magnification objectives as in the
EXAMPLE below:
Diameter of the field using X10 objective = 2 mm
Diameter of the field using, X40 objective = 2 x 10/40 mm = 0.5 mm
1.2. MATERIALS
Pasteur pipettes
methylene blue
ethanol
isopropanol (to
light microscope
hemocytometer
microscope slides
scalp hair
coverslips
elodea leaves
clean objective
safranin stain
lenses)
and bulbs
1.3. PROCEDURE
1.3.1.Measurement of scalp hair diameter
1. Measure the visual field diameter with a hemocytometer. You should use both 10X
and 40X objective lenses. Edge of the smallest square in the hemocytometer is 0.05
mm. Compare the two obtained values. Is there a meaningful proportion?
2. Clean a new glass microscope slide and coverslip with ethanol and tissue paper.
3. Pull out one hair from your scalp. Cut a piece shorter than the coverslip and place on
the slide in the center.
4. Place a drop of water than the coverslip on the hair.
5. Observe your sample using 40X objective lens. To estimate the thickness of the
sample, line up one edge of the hair against the edge of the visual field. Estimate the
proportion occupied by the hair.
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1.3.2. Observation of Elodea Leaf Cells
Young leaves at the growing tip of elodea are particularly well suited for studying cell
structure because these cells are only a few cell layers thick.
1. With a forceps, remove a single young leaf, mount it in a drop of water and cover with
a coverslip.
2. Examine under 40X.
3. Add a drop of safranin stain to make the cell wall more obvious. Add the stain (1
drop) to one edge of the coverslip then draw the stain under the coverslip by touching
a piece of tissue paper to the opposite site of the coverslip.
1.3.3. Observation of Epithelial Cells
1. Scrape the inside of your cheek with a flat toothpick.
2. Drop a methylene blue on a slide and stir the scraped epithelial cells in the stain.
3. Examine the preparation under 40X magnification.
4. Draw the two cell types you have examined and state the obvious differences between
them.
1.4. QUESTIONS
1. What are the main differences between bright and dark field microscopy? Which type of
samples better suit for bright and dark field microscopes, respectively?
2. Which part of the cell does safranin and methylene blue stain?
3. What are your estimates for the diameter of elodea and epithelial cells? Are these values
consistent with the known ones?
1.5. REFERENCES
1. Figure 1.1:
http://www.lmpc.edu.au/resources/Science/research_projects/light_microscope/light_micro
scope.htm
2. Background information:
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.html
3. Figure 1.3, modified from:
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html
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EXPERIMENT NUMBER: 2
“PLASMA MEMBRANE”
2.1. INTRODUCTION
The plasma membrane serves as the interface between the machinery in the interior of
the cell and the extracellular fluid that surrounds all cells. It consists of a phospholipid bilayer
together with proteins that can span the bilayer (integral/ transmembrane) or are peripherally
attached to one face or the other (peripheral). The components of the bilayer frequently move
laterally into other regions of the membrane, making it appear more fluid than static. This is
called the fluid mosaic model of the cell membrane. In addition, proteins on the extracellular
face can be heavily glycosylated, giving the membrane asymmetrical characteristics.
The plasma membrane is selectively permeable; that is it will allow some substances
to pass across it but not others.
Molecules and ions move spontaneously down their concentration gradient (i.e., from
a region of higher to a region of lower concentration) by diffusion.
Molecules and ions can be moved against their concentration gradient, but this
process, called active transport, requires the expenditure of energy (usually from
ATP).
Most membranes are permeable to water. Osmosis is a special term used for the
diffusion of water through cell membranes.
When the concentration of the solute is the same on the inside and outside of the cell,
the water moves equally in both directions. The solution outside the cell is called
isotonic. If the solution contains a higher solute concentration than the cell, it is
hypertonic, but if it is lower, the solution is hypotonic.
2.2. MATERIALS
light microscope
starch solution
NaCl soln.s
cellophane tube
iodine solution
lancet
tube clips
Elodea leaf
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2.3. PROCEDURE
2.3.1. Selective Permeability of an Artificial Membrane (CELLOPHANE)
1. Close one end of the cellophane tube with a clip.
2. Fill the tube about 3/4 full with starch solution.
3. Squeeze out the air and clamp the top with a second clip.
4. Wash the bag to remove any starch from the outside and blot dry with a tissue.
5. Submerge the bag in a beaker of iodine solution. Note the color of the bag and the color of
the iodine solution.
6. After 30 minutes, remove the bag, rinse in water and blot dry. Note the color of the bag,
and the iodine solution.
7. Report and explain your observations.
2.3.2. Osmosis in Elodea Leaf Cells & Human Blood Cells
Slide 1:
Sample: elodea
Sample: elodea
Solution added: none
Solution added: 0.1M NaCl
Slide 2:
Sample: elodea
Sample: elodea
Solution added: 0.3M NaCl
Solution added: 0.6M NaCl
Slide 3:
Sample: blood
Sample: blood
Solution added: isotonic salt soln.
Solution added: water
Slide 4:
Sample: blood
Sample: blood
Solution added: 0.3M NaCl
Solution added: 0.6M NaCl
Slide 5:
Sample: elodea
Sample: blood
Solution added: unknown soln.
Solution added: unknown soln.
Figure 2.1. Split slides for Elodea and blood cells
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1. Take 5 microscope slides, draw a perpendicular line dividing each slide into two parts
and label the all 10 parts according to the sample and the solution to be added (Figure
2.1) with a permanent marker.
2. Observe them under 40X magnification 5 min after the solutions are added.
3. Note which solution induces shrinking or swelling. You should define the solutions in
terms of hyper-, hypo- and isotonicity.
2.4. QUESTIONS
1. Can you estimate the concentration of XM NaCl?
2. Please define selective permeability of the plasma membrane.
3. What other functions can be attributed to the plasma membrane?
2.5. REFERENCES
1. Background information: http://darwin.nmsu.edu/~molbio/cell/PM.html
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EXPERIMENT NUMBER: 3
“CELLULAR FRACTIONATION”
3.1. BACKGROUND INFORMATION
Each organelle has characteristics (size, shape and density for example), which make it
different from other organelles within the same cell. If the cell is broken open gently, each of
its organelles can be subsequently isolated. The process of breaking open cells in an isotonic
buffer is homogenization and the subsequent isolation of organelles is cellular fractionation.
Isolating the organelles requires the use of physical chemistry techniques, and those
techniques can range from the use of simple sieves, gravity sedimentation or differential
precipitation, to ultracentrifugation of fluorescent labeled organelles in computer generated
density gradients.
In this experiment, cellular fractionation of a homogenized rat liver will be
accomplished via a technique called differential centrifugation, which depends on the
principle that as long as they are denser than the surrounding medium, particles of different
size and shape travel toward the bottom of a centrifuge tube at different rates when placed in a
centrifugal tube. Particles with higher density will sediment at a faster rate than the less dense
ones.
The first steps of differential centrifugation do not generally yield pure preparations of
a particular organelle; so further steps are usually required. In many cases, further purification
is accomplished by centrifugation of crude extract through sucrose density gradients.
There are two types of centrifuges according to their rotors (centrifuge heads):
Swinging-bucket model: rotors allow the tubes to swing out, causing the particles to
move in a direction parallel to the walls of the tube.
Fixed-angle model: the tubes are maintained at a particular angle, so that the particles
sediment not to the bottom, but to the wall of the tube.
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Relative centrifugal force (RCF) is defined as the ratio of the centrifugal force to the
RCF = Fc/Fg = ω2r/980a
force of gravity, or
π (expressed in radians/second) is converted to revolutions per minute (rpm) by substituting
RCF = 1.119 x 10-5 (rpm)2r
ω = π (rpm) / 30, resulting in;
where r is expressed in cm. RCF units are expressed as “g”.
3.2. MATERIALS
Fresh rat liver
0.25 M sucrose
High-speed
50 ml centrifuge
centrifuges
tubes
Table-top and
Vortex
Homogenizer
Micropipettes and
tips
Ice bucket
3.3. PROCEDURE (Figure 3.1)
1. Chop rat liver into approximate few mm3 pieces.
2. Add 0.25 M sucrose (10% w/v).
3. Homogenize with hand blender.
4. Centrifuge the homogenate to remove the cell debris at 800 g, 5 min.
5. Collect the supernatant: this is your whole homogenate. Save 5 ml for the next experiment
(Tube H) and record the volume of the rest.
6. Centrifuge the rest of the homogenate for 15 min at 5,000 g.
7. Resuspend the nuclear pellet in 0.25 M sucrose (save the suspension, Tube #2).
8. Centrifuge for 10 min at 24,000 g.
9. Resuspend the mitochondrial pellet in 0.25 M sucrose (save the suspension, Tube #3).
10. Rename Tube #2 as Tube N andTube #3 as Tube M.
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3.4. QUESTIONS:
1. Why was “liver” chosen as the target organ for cellular fractionation?
2. Why was homogenization and subsequent suspension of organelles done in 0.25M
sucrose solution?
3. Why were all steps carried out in relatively lower temperatures (centrifuges were fixed
to 4ºC and organelles were stored at -70ºC)?
4. Please calculate the radius for centrifuge #1 using the given “g value” in the manual
and the “rpm value” read from the centrifuge.
5. Please calculate the “rpm values” for centrifuge #2 using the given “g and r values”
given in the manual.
6. Why do you think that the organelles obtained with this method are not absolutely
pure? Can you suggest an additional or alternative method to better purify these
organelles?
3.5. REFERENCES
1. Background information: http://homepages.gac.edu/~cellab/chpts/chpt3/intro3.html
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homogenized rat liver
in 0.25M sucrose
Tube #1
Centrifuge#1
800g
5 min
Tube #1
Cell Debris
Collect supernatant into a new tube
Tube #2
save 5 ml* (Tube H)
“Whole Homogenate”
5,000g
15 min
Centrifuge#2
r=9.4cm
Tube #2
Nuclear Pellet
(a)
Collect supernatant in a new tube
Centrifuge#2
24,000g
r=9.4cm
(b)
Resuspend**
10 min
Tube #2
“Nuclear Suspension”*
Tube #3
Mitochondrial Pellet
(a)
Resuspend**
Tube #3
“Mitochondrial Suspension”*
Figure 3.1. Schematic diagram of differential centrifugation
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EXPERIMENT NUMBER: 4
“ANALYSIS OF SUBCELLULAR FRACTIONS I”
4.1. INTRODUCTION
4.1.1. Measurement of Succinate Dehydrogenase Activity
Mitochondria enclose the biochemical machinery for cellular respiration; the aerobic
processes by which sugars, fatty acids, and amino acids are broken down to carbon dioxide
and water and their chemical energy captured as ATP. The Krebs cycle (also called as
tricarboxylic acid or citric acid cycle) is a key series of reactions in this aerobic process
(Figure 4.1). One of the best-studied enzymes in Krebs cycle is succinate dehydrogenase
(SDH), which catalyzes the following redox reaction:
Succinate + FAD Fumarate + FADH2
The objective of this lab is to measure the rate of the reaction in vitro using the
mitochondrial fraction isolated in the previous lab session.
The succinate → fumarate reaction is measured by monitoring the reduction of an
artificial electron acceptor, DCIP. DCIP is supplied as a dark blue solution and the color of
this solution becomes lighter as DCIP is reduced. As the solution becomes lighter the
absorbance at 600 nm decreases gradually which is observed via a spectrophotometer.
To use an artificial electron acceptor, the normal path of electrons in the electron
transport chain (ETC) must be blocked. This is accomplished by adding sodium azide to
the reaction mixture. This poison inhibits the transfer of electrons from cytochrome a3 to the
final acceptor, oxygen, so that electrons cannot be passed along:
SDH
Succinate + FAD → Fumarate + FADH2
FAD + 2H
e-
+
Electrochemical
gradient
DCIP
blue
Azide
ETC
colorless
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Figure 4.1. The Krebs cycle
4.1.2. Spectrophotometry
Spectrophotometry is the measurement and analysis of electromagnetic radiation
absorbed, scattered, or emitted by atoms, molecules, or other chemical species. The
spectrophotometer operates by passing a beam of light through a sample and measuring the
intensity of light reaching a detector.
The UV-Vis spectrum is from 200 nm to 800 nm. UV-Vis Spectrophotometry uses
ultraviolet and visible electromagnetic radiation to energetically promote valence electrons in
a molecule to an excited energy state. The UV-Vis spectrophotometer then measures the
absorption of the energy to promote the electron by the molecule at a specific wavelength or
over a range of wavelengths. The amount of electromagnetic radiation absorbed by a species
23
in a solution (A) depends on its concentration (c), the path length of the electromagnetic
radiation (b), and the specific molar absorptivity (e) of the species:
Beer-Lambert's Law A = ebc
The absorption of energy also depends upon the intensity of the incident light, Io, and
the intensity of the exiting electromagnetic energy, I, where:
A = log (I0 / I)
There are five major components to a UV-Vis spectrophotometer; a radiation source, a
wavelength selector or monochromator, a sample cell or cuvette, a detector, and a readout
device.
Figure 4.2. The mechanism of spectrophotometer
“Transmittance (T)” is another term that implies the amount of light energy that is
transmitted.
%T = T x 100 A = - log10T
Cuvettes, or cells that contain the sample to be analyzed, should have parallel sides
that are perfectly perpendicular to the radiation source. The cuvette used in UV-Vis
spectrophotometry usually has a path length of 1 cm. The cuvette can be made up of several
materials including fused silica, quartz, and plastic. Quartz cuvettes are transparent to UV
and visible radiation and therefore are more commonly used for UV-Vis spectrophotometry.
The cuvette must be wiped free of any marks or fingerprints before scanning. Any marks on
the cuvette can scatter the UV-Vis radiation and cause error in the experiment. After each
24
scan the cuvette should be cleaned thoroughly. While scanning, the compartment holding the
sample cell must be closed to all outside light sources. This prevents error in the experiment.
4.2. MATERIALS
Mitochondrial fraction, “Tube M” from previous lab session
Assay medium
Malonate (0.2 M)
micropipettes
Sodium azide
Succinate (0.2 M)
ice bucket
plastic cuvettes
spectrophotometer
(0.04M)
DCIP (5 x 10-4M)
4.3. PROCEDURE
4.3.1. Measurement of Succinate Dehydrogenase Activity
1. Set the wavelength (λ) of the spectrophotometer at 600 nm.
2. Label 10 tubes as shown in Table 5.1. Except for the ice-cold mitochondrial
suspension (MS), all solutions should be at room temperature.
3. Prepare the MS for tube 7 by heating in microwave oven and then cooling on ice.
4. To all tubes, add the various solutions given in Table 4.1, except for MS in the stated
order:
Table 4.1. Contents of tubes to be prepared
a
Tube
Assay Medium
(ml)
Azide
(ml)
Order
1st
2nd
3rd
Blank1
3.7
0.5
1
3.2
Blank2
Succinate
(ml)
MSa (ml)
4th
5th
6th
-
-
0.5
0.3
0.5
0.5
-
0.5
0.3
3.1
0.5
-
-
0.5
0.9
2
2.6
0.5
0.5
-
0.5
0.9
Blank3b
3.4
0.5
-
-
0.5
0.6
3
2.9
0.5
0.5
-
0.5
0.6
4
2.7
0.5
0.5
0.2
0.5
0.6
5
3.4
-
0.5
-
0.5
0.6
6
3.4
0.5
0.5
-
-
0.6
7
2.9
0.5
0.5
-
0.5
0.6c
DCIP (ml) Malonate (ml)
Mitochondrial Suspension. bBlank3 serves as blank solution for both tube 3 and tubes 4,5,6 and 7. cThe MS
to be added to tube 7 will be heated
25
5. Add MS to Tube #1 and take the absorbance readings at t0. Repeat this step for each
tube (Add MS and read the absorbance one by one for each tube, so that the
absorbance reading can be obtained for t = 0 min).
Tube 1
Blank1
Tube 2
Blank2
Tubes 3-7
Blank3
6. At 5 min-intervals, measure the absorbance of the seven tubes for 35 min. Always
remember to adjust for the 3 blanks as in step 6. Record all absorbance readings on
DATA SHEET 1 (for t0, 5, 10, 15, 20, 25, 30 and 35).
4.4. QUESTIONS
1. On DATA SHEET 2, enter for each tube the total change in absorbance (∆A) at each time
interval.
Absolute values !
∆A(Tube n) = │A(Tube n at ti) – A(Tube n at t0)│
2. Plot the reaction rates for tubes 1-6, in other words, ∆A versus time (t0
to 35).
You may
either plot these 6 curves (not linear, draw as curves!) on a single graph or on 6 separate
graphs.
3. Plot the initial velocity (Vi) versus enzyme concentration ([E]) for tubes 1-3 (a single
curve, which starts from the origin).
Vi (∆A/min) = │ A(Tube n at t35) – A(Tube n at t0)│/ ∆t
[E]for each tube = VolumeMS added
4. Comment on what happens to Vi when [E] has doubled and tripled.
5. What volume of mitochondrial suspension gives the highest initial velocity? Explain.
6. In tubes 4, 5, 6 and 7, four different factors that affect the rate of this reaction are used.
State the nature of these factors and discuss their observed and expected effect on the
reaction.
7. In the electromagnetic spectrum, where does the light with a λ of 600 correspond? Why do
you think this λ was selected? Why was the absorbance reduced as the reaction preceded?
8. Malonate is a competitive inhibitor of SDH. What is the action mechanism of a
competitive inhibitor in general? Do your results support this statement?
9. Krebs cycle contains a variety of enzymes. Why do we choose SDH, but not another
enzyme for this experiment?
NOTE: Include the data sheets and graphs in the results part.
References:
1.
Background: http://www.easternct.edu/personal/faculty/adams/Resources/Lab3%20SDH.pdf
2. Figure 4.1: http://www.personal.kent.edu/~cearley/PChem/pchem.htm
26
EXPERIMENT NUMBER: 5
“ANALYSIS OF SUBCELLULAR FRACTIONS II”
5.1. INTRODUCTION
5.1.1. Nucleic Acid To Protein Ratio
Many molecules display maximum absorbance (Amax) at a particular wavelength (λ). This
property may be used in identification of molecules.
For nucleic acids; all four bases of DNA, accordingly the whole DNA molecule
absorbs at λ = 260 nm. This Amax is used to determine the amount of both DNA and RNA.
For proteins; Amax is reached at λ = 280 nm provided that the protein contains a normal
percentage of tyrosine and tryptophan amino acids (aromatic rings). Proteins also show
another Amax at a smaller λ in UV, due to their peptide bonds.
In this experiment, the A260 values of the fractions collected by differential centrifugation
will be measured. Assuming that “An A260 of 1 is equivalent to 50 µg/ml dsDNA”, the DNA
concentration in each fragment will be estimated.
5.1.2. Microscopic Examination Of Nuclear Fraction
The nuclear fraction that has been isolated in the previous experiment (Tube N) will be
examined microscopically to identify the nuclei, in addition approximate size of the nuclei
will be measured (refer to Experiment #1).
The nucleus is separated from the cytoplasm by an envelope consisting of two
membranes. The entire chromosomal DNA is held in the nucleus; packaged into chromatin
fibers by its association with an equal mass of histone proteins. The nuclear contents
communicate with the cytosol by means of openings in the nuclear envelope called nuclear
pores. Nucleoli are large, round or oval structures, in which ribosomal subunits are
assembled, thus are rich in RNA and protein.
27
For the observations on nuclei, the stain to be used is aceto-orcein, which stains
chromatin red. The nucleoli stand out since they do not stain with the orcein. Each nucleolus
appears as a prominent, round, clear area.
5.2. MATERIALS
Spectrophotometer
All fractions from
Light microscope
Experiment #3
Micropipettes
Aceto-orcein
5.3. PROCEDURE
5.3.1. Determination of DNA Concentration
1. Thaw all fractions and mix well.
2. Prepare 18 tubes and label as follows:
H1, H2, H3, H4, H5, H6
→
for whole Homogenate
N1, N2, N3, N4, N5, N6
→
for Nuclear pellet
M1, M2, M3, M4, M5, M6
→
for Mitochondrial pellet
3. Prepare serial dilutions of each fraction by increasing the dilution ratio by 5.
Table 5.1. “Serial Dilution” steps for each fraction
Tube
0.25 M
Sucrose(
ml)
Previous tube (ml)
1
Volumefinal (ml)
Final dilution
ratio
1.25 ml Stock Solution
Volume left after
dilution (ml)
1.00
2
1
0.25
1.25
1/5
1.00
3
1
0.25
1.25
1/25
1.00
4
1
0.25
1.25
1/125
1.00
5
1
0.25
1.25
1/625
1.00
6
1
0.25
1.25
1/3125
1.25
28
4. Measure the absorbance values (A260) of all 24 tubes and determine the dilution factor for
each fragment, which gives an absorbance between 0.2 and 1.5.
5. Try to calculate the DNA amount in each fraction by taking the dilution factors into
account.
DNA concentration (µg/ml) = A260 x 50 µg/ml x dilution factor
5.3.2. Microscopic Examination Of The Nuclear Fraction
1. Smear a tiny amount of the nuclear pellet on a clean slide with a spatula.
2. Immediately before the smear dries, add several drops of lacto-aceto-orcein. After 15
sec add a cover slip and press out the excess stain with a paper towel very gently.
3. Examine the preparation under the medium power objective. After locating a region
with nuclei, switch to the high power objective.
4. Draw a typical nucleus, labeling nucleolus and estimate the nuclear diameter (refer to
EXPERIMENT 1).
5.4. QUESTIONS
1. What does endosymbiotic theory suggest?
2. What was the reason that we have used plastic cuvettes in Experiment #4 and quartz
cuvettes in this experiment?
3. The spectrophotometers cannot measure absorbance values higher than 2. Why?
29
EXPERIMENT NUMBER: 6
“EXTRACTION OF DNA FROM BOVINE SPLEEN”
6.1. INTRODUCTION
6.1.1. DNA Extraction
Isolation of nucleic acids is the first step in most molecular biology studies. Extraction
of nucleic acids from biological material requires cell lysis, inactivation of cellular nucleases
and separation of the desired nucleic acid from cellular debris. Common cell lysis procedures
include; Mechanical disruption (ex. grinding, hypotonic lysis), Chemical treatment (ex.
detergent lysis) and Enzymatic digestion (ex. Proteinase K).
The extraction medium usually contains an ionic detergent, which is required to lyse the
nuclei and release the DNA. The detergent also inhibits any nuclease activity present in the
preparation. Combination of phenol-chloroform and high concentrations of salt are often used
to eliminate contaminants from nucleic acids. After cell lysis and nuclease inactivation,
cellular debris may easily be removed by precipitation. Nucleic acids are usually precipitated
with isopropanol or ethanol.
6.1.2. Gel Electrophoresis
Nucleic acids may be separated electrophoretically on gel systems according to their size,
shape and overall charge density (charge per unit of mass). This separation is commonly done
on horizontal agarose gels. Due to their negatively charged phosphate backbone, nucleic acids
move towards the anode in the electrical field. In the presence of ethidium bromide (EtBr),
the separated nucleic acids are visualized under UV light. EtBr intercalates between the two
strands of DNA. Electrophoresis is frequently used to determine size and purity of DNA.
6.1.3. Spectrophotometry
The ratio of absorbance at λ = 260 to λ = 280 nm is one measure of the purity of a
nucleic acid preparation. The 260/280 ratio of purified DNA is about 2. Higher ratio is often
due to RNA contamination and lower values to protein contamination.
6.2. MATERIALS
Bovine spleen
Chilled blender
Agarose
Table-top centrifuge
Saline Citrate Buffer
0.5X TEB
Spectophotometer
2.6M NaCl
10X Loading buffer
quartz cuvettes
Absolute ethanol
30
6.3. PROCEDURE
6.3.1. DNA Extraction
homogenized bovine spleen (15g) in
150 ml cold SSC buffer dispersed
in 4 centrifuge tubes
4,000 rpm
Materials soluble in physiological
buffer (ex: RNA, many
carbohydrates, some proteins)
15 min, 4ºC
DNP (DNA + Proteins), cell debris,
unbroken cells
Add 40 ml of 2.6 M NaCl to each centrifuge tube.
Vortex shortly to aid dissociation of the pellet.
Shake vigorously till all pellet is dissolved.
SALTING OUT
5,000 rpm
DNA dissolved in
aqueous medium
(salt solution)
20 min, RT
Proteins dissociated from DNA, cell
debris, unbroken cells
Combine the supernatant of all 4 tubes in one beaker
Add 2X volume of absolute Ethanol
DNA: fish out with a sterile tip, air dry
then incubate overnight at room
temperature in Buffer TE (Tris-EDTA).
Store at -20ºC.
31
Sections after 6.3.2. will be performed in the next lab!!!
6.3.2. DNA analyses with electrophoresis and spectrophotometry
Agarose Gel Electrophoresis
1. Prepare 1% agarose (w/v) in 35 ml of 0.5X TEB. Boil in microwave oven and let cool
till about 55ºC.
(Calculate: .......... g of agarose in 35 ml of 0.5X TEB)
2. Add 1.5 µl Ethidium Bromide (EtBr) to agarose solution, mix and pour into the gel
tray.
(CAUTION!! EtBr is a potential carcinogen, do not touch or inhale!).
3. Let it polymerize for 10 min.
4. Prepare serial bovine DNA dilutions of 1:10, 1:100 and 1:1000.
5. Mix the samples (a control sample provided by the assistant and serial bovine DNA
dilutions) with 6X loading buffer (glycerol + xylene cyanol + bromophenol blue) in
5:1 ratio on a piece of Parafilm and load the samples into sample wells. Run the gel
for about 10 min.
6. Visualize the gel under UV light and record to the computer.
Notice the differences between the mobility patterns of control and your samples. Try
to explain this difference.
Spectrophotometry
1. Calculating the A260/A280 ratio will check the purity of the control and bovine DNA
(Use 1:100 dilution of both control and bovine DNA).
2. Calculate the concentrations of both control and bovine DNA (refer to Experiment 5).
6.4. QUESTIONS:
1. Please explain the mechanism of salting out?
2. Can you suggest a method other than salting out to separate DNA from proteins?
32
EXPERIMENT NUMBER: 8
“MITOSIS AND CYTOKINESIS”
7.1. INTRODUCTION
Recall that there are two basic cell types; prokaryotic and eukaryotic. Because of their
simple genetic material, prokaryotes reproduce primarily as a result of fission; the splitting of
a precasting cell into two, with each new cell receiving a full complement of the genetic
material.
In eukaryotes, the process of cell division is more complex, primarily because of the
much more complex nature of the hereditary material, DNA (deoxyribonucleic acid) and the
proteins complexed to it. In these cells, the genetic material is organized into chromosomes.
Cell division usually involves 2 processes; mitosis (nuclear division) and cytokinesis
(cytoplasmic division). Whereas mitosis results in the production of two nuclei, both
containing identical chromosomes, cytokinesis ensures that each new cell contains all the
metabolic machinery necessary for sustenance of life.
Dividing cells pass through a regular sequence of events called the cell cycle. The
majority of the time is spent in interphase and actual nuclear division-mitosis- is only a brief
portion of the cycle. During interpase, the cell is scanning its environment for growth factors,
producing new DNA, assembling proteins from amino acids, and synthesizing or breaking
down carbohydrates. In short, interphase is a busy time in the life of a cell.
7.1.1. Mitosis in Onion Root Cells
Cell divisions in plants localized in specialized regions called meristems. Meristems
are regions of active growth. Plants have 2 types of meristems: apical and lateral. Apical
meristems are found at the tips of plant organs (shoots and roots).
33
Figure 7.1. Stages of mitosis
7.2. MATERIALS
light microscope
previously prepared slide of onion root cells
7.3. PROCEDURE
Obtain a prepared slide of a longitudinal section of an Allium (onion) root tip. This
slide has been prepared from the terminal part of an actively growing root. It was "fixed" by
chemicals to preserve the cellular structure and stained with dyes that have an affinity for the
structures involved in nuclear division.
Observe and draw cells undergoing different stages mitosis.
Try to see the distinctive features of each stage.
34
Try to estimate the number of chromosomes of this organism.
Find a cell undergoing cytokinesis. State how cytokinesis takes place in this tissue.
7.4. QUESTIONS
Part I
1. Which part of the root was chosen for observing mitosis? Why?
2. Please estimate chromosome number for onion cells (2n=?). You should estimate this
value through counting the number of chromatits observed in the metaphase plate or
migrating in anaphase.
3. Which type of cyokinesis takes place in these cells?
Part II
1. Intensity of DNA band is directly proportional to DNA concentration. Explain if your
results are consistent with this fact.
2. Do you think that the bovine DNA is contaminated according to the A260/A280 ratio? If
so, what may be the reason?
References:
Figure 7.1 and background information:
http://www.accessexcellence.org/RC/VL/GG/mitosis.html
35
EXPERIMENT NUMBER: 8
“ANIMAL CELL CULTURE”
8.1 INTRODUCTION
8.1.1 What is cell culture?
The technique that is used to grow eukaryotic or prokaryotic cells in vitro (Latin: in glass) is
called “cell culture”. Even though the term refers to a much larger scale of applications, in
practice it almost exclusively refers to “animal cell culture” techniques.
Since its establishment, animal cell culture techniques have allowed scientists to solve many
puzzles on animal cell biology by allowing them to work under very strictly defined and
controlled conditions. Moreover, its rapidity and cost efficiency together with complete
control over the experiments makes this technique invaluable for scientists.
Animal cell culture techniques are routinely applied in many laboratories today and allow the
use of many applications formerly inapplicable or only possible by in vivo experiments. Such
applications include:
•
•
•
Production of monoclonal antibodies
Gene knock-out and over-expression studies
Production of valuable proteins (i.e. enzymes and hormones)
Two basic findings lie in the core of cell culture technique are:
1. Cells can continue their growth under in vitro conditions as they can in their original tissues (as
first shown by Ross Harrison in 1907[1]).
2. Cells can be frozen and stored for long periods of time and can still be viable after thawing.
Cell culture, very roughly speaking, consists of growing cells in vitro (plastic dishes),
harvesting cells from plates, splitting and seeding cells on fresh plates and storing them by
freezing when necessary (preferably in liquid nitrogen: -196o C).
Animal cells are much more “demanding” than prokaryotic cells and thus their maintenance
requires tedious and careful work, carefully chosen and prepared solutions and equipment,
sterile and well conditioned environment as well as a carefully designed culture medium that
mimics in vivo conditions. Selection of appropriate cell type among many is also crucial for
the outcome of the experiment.
8.1.2 Source of Cells
Primary cells: The cells are directly taken from the target tissues of organisms and added to
the culture medium.
Continuous cell lines: They are well established stable cell lines, mostly isolated from
cancerous tissues. There are also some cell lines that are produced by manipulating their
genome of a primary cell to overcome senescence; these cells are called “immortalized”.
8.1.3 Media
In biology, the media used for various purposes can be classified into two categories:
Defined media: The composition of the medium is known (i.e. which compounds are present
and what are their concentrations)
Undefined media: The composition of the medium is not known. Those media contain a
component whose composition is not exactly known. For example the composition of yeast
36
extract, a component of Luria-Bertani (LB) broth, is unknown and thereby it renders LB broth
undefined eventhough the exact composition of other ingredients are known.
The most commonly used formula of an animal cell culture medium contains a “basal
medium” and some “other components” that support the cell growth.
There are defined and undefined media recipes for cell culture. Defined media for cell culture
contains only basal media, whose composition is known; where as undefined media contains
basal media and serum. Fetal bovine serum (FBS) is the most common type of serum used in
cell culture and it is obtained from the fetuses of cows thus its composition is not exactly
known.
Basal media
The selection of basal medium is crucial for the establishment of a healthy and well growing
cell culture. Different cell types require different composition of substances and therefore
different kinds of basal media for their optimal growth. There are many basal media types,
such as Eagle’s medium and its derivatives (EMEM, AMEM, DMEM, GMEM, JMEM),
Rosewell Park Memorial Institute medium derivates (RPMI 1629, RPMI 1630, RPMI 1640),
Fischers’s, Liebovitz, Trowell, and William’s media, CMRL 1060, Ham’s F10 and
derivatives, TC199 and derivatives, MCDB and derivatives, NCTC and Waymouth [2].
Components of basal media [2]
1. Balanced salt solution: They contain inorganic salts to maintain physiological pH and osmotic
pressure, and to provide necessary ions that are used for key metabolic activities (membrane
potential, cofactors) (Na+, K+, Mg2+, Ca2+, Cl-, SO44+, PO43+, and HCO3-).
2. Buffering system: Mostly used media are buffered with bicarbonate ions. HCO3- ions react
with CO2 formed by the cells through oxidative respiration and also with CO2 supplied in the
atmosphere. There are different buffering systems which use high concentrations of PO43+ or
beta-glycerophosphate and low bicarbonate. It is also possible to use organic buffers such as
HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid).
3. Energy source: Glucose. Other sugars can be used (i.e. maltose, sucrose, fructose, galactose
and manose).
4. Amino acids
5. Vitamins
6. Hormones and growth factors (they are normally present in serum but in defined media they
are added to the basal medium)
7. Proteins and peptides
8. Fatty acids and lipids
9. Accessory factors (zinc, iron, copper, selenium)
10. Antibiotics
Serum [2]
1.
2.
3.
4.
5.
Growth factors
Albumin
Transferrin
Anti-proteases
Attachment factors
8.1.4 HEK 293T cell line
HEK 293T – or simply 293T – cell line is a highly transfectable derivative of human
embryonic kidney 293 cells. 293 cells were obtained from and aborted fetus’ kidney cells by
37
Frank Graham in late 70s [3,4]. Those cells were transfected with sheared adenovirus 5 DNA.
At first the effect of this transfection at the genomic level was unknown, but it was observed
that these cells overcame senescence, in other words “immortalized”. In 1997, Nathalie Louis
and her colleagues found out that approximately 4.5 kilobases of DNA from the left arm of
the viral genome was inserted into chromosome 19 [5]. In addition to adenovirus 5 DNA,
293T cells are also transfected with simian virus 40 (SV40) large T antigen is incorporated
into the genome [6].
8.1.5 Reporter Gene
Reporter gene is basically a gene that produces a detectable product or observable effect when
introduced into the cell. Reporter genes are generally attached to other genes of interest and
used to see if the transfection procedure has been successful or to measure the activity of a
specific gene. Green fluorescent protein (GFP), luciferase and β-galactosidase are common
examples.
8.1.6 Vector
Vectors are “molecular carriers” that are used to introduce foreign DNA into the prokaryotic
or eukaryotic cells. Plasmids and viruses are two types of vectors used in molecular biology.
Plasmid vectors contain four important sites:
•
•
•
•
An origin of replication
A genetic marker
A multiple cloning site
Promoter site(s)
Every plasmid has to have an origin of replication since it is required for plasmid, and thereby
the DNA of interest that is inserted into the plasmid, to replicate.
Genetic markers allow us to select the successfully transfected cells. Antibiotic resistance
genes are commonly used in cell culture as genetic markers.
The DNA fragment of interest is inserted into the multiple cloning site. This site contains
many recognition sites for different restriction enzymes thereby allowing us to use a suitable
enzyme for our experiment.
In eukaryotes, the promoter sequence is a part of gene regulation. For a gene to be transcribed,
the appropriate transcription factor must bind to the promoter region and “turn the gene on”.
Different promoters differ in activity; some promoters are highly active (i.e. CMV) where as
some are not. It is possible to construct plasmids with different promoter regions and thereby
altering its activity.
38
Figure 8.1. Map of pcDNA3 from Invitrogen Life Technologies.
8.1.7 Green Fluorescent Protein
Green fluorescent protein is isolated from the jellyfish Aequorea Victoria. Thanks to its
unique shape, this protein fluoresces green when excited by blue light. GFP is 238 amino
acids length and has a unique structure comprised of 11 β-barrel and a single alpha helix
which contains the chromophore [7, 8]. To visualize GFP a special type of microscope,
namely “inverted fluorescent microscope” is used. This microscope is capable of exciting the
cells with light at specific wavelengths by using special filters and visualizing fluorescence
emitted by the cells. Its light source and lenses are inverted for better focusing.
Figure 8.2. An inverted light microscope.
39
Introducing foreign DNA into cells
There are various techniques to introduce foreign DNA into cell. These techniques include
calcium phosphate, electrophoration, heat shock, transduction using viruses that are incapable
of replicating, via commercial transfection reagents etc.
For the introduced DNA to be translated, it has to enter the nucleus. The majority of the
foreign DNA is degraded and only a small proportion reaches the nucleus. In the nucleus, it
may stay free in the plasmid form or it may be inserted into the host’s genome. If it remains
free as a plasmid it is called a “transient transfection” because the transfected DNA can be
lost as the cells proliferate. If the transfected DNA is inserted into the host’s genome it is
called a “stable transfection” and it will remain in the host’s genome regardless of
proliferation.
8.2. MATERIALS
6 well culture plates (Figure 8.3), Inverted Microscope (Figure 8.2)
Plasmid DNA pEGFP-C2
Micropipets, Sterile tips and tubes (Autoclaved)
DMEM (Dulbecco/Vogt Modified Eagle's Minimal Essential Medium)
2M CaCl2, 2X HEPES Buffer
8.3. METHODS
1. Start with 293T cells cultured on 6 well plates (0.8x106 cells/well) with 1 ml of DMEM
added.
2. Prepare the transfection solutions in 6 tubes (1.5ml centrifuge tubes) as indicated on Table
8.1 and in Figure 8.3. Wells 1 and 2 will be negative controls; add H2O instead of plasmid
DNA to well 1 and add empty plasmid (i.e., plasmid with no reporter gene) to well 2. You
will add 0.1 µg plasmid to wells 3 & 4 and 1 µg to wells 5 & 6, respectively.
3. Mix the tubes by tapping after addition of both CaCl2 and HEPES.
4. Wait growth of DNA/Ca-P particles for 5 min after all contents of the transfection solution
is mixed.
5. After 5 min, spread the DNA/Ca-P onto the wells by pipeting all over each well.
6. Incubate cells with transfection solution at 37°C overnight.
7. Next Day Evaluation of transfection with inverted microscope
Inverted Microscope at AKIL Lab, BE ON TIME !
Check and take picture of your cells both with and without florescence filter.
- Without Filter all cells are visible
- With Filter only cells transfected with pEGFP-C2 is visible.
Table 8.1. Components of transfection solutions
H2 O
H2O or Plasmid DNA
2M CaCl2
2X HEPES
Total Volume
Spread on each well
for 6 well plates (solutions in µl)
for 1 well
209.5
10.0
30.5
250.0
500.0
500
40
Figure 8.3. Schematic representation of a 6-well culture plate. Wells are numbered and
labeled according to the kind of plasmid DNA added.
Questions
1. What is the advantage of using in vitro techniques than in vivo techniques?
2. Why does the transfected DNA have to enter the nucleus to be translated?
3. Why the selection of the appropriate cell type (e.g. kidney cell, fibroblasts, neuronal cells etc) is
crucial for the experimental design and results
4. Please shortly explain two of the transfection methods (you can use the ones mentioned in the lab
manual or find different ones in the books/ on the internet).
5. What is a defined medium? Undefined medium? Why it is preferred to use defined media instead of
undefined media?
Bonus
1. Eukaryotic cells, like prokaryotic cells, have a defense mechanism against the foreign DNA.
They recognize and destroy any piece of DNA if it is detected as not of the cell’s own. Even
though in the cell lines used for transfection this defense mechanism is intact, it is still
possible to successfully introduce foreign DNA into the cell. Propose a rational explanation
for this phenomenon.
References
[1] Harrison, R. G. (1907). Proc. Soc. Exp. Biol. Med., 4, 140
[2] Basic Cell Culture: A Practical Approach. J. M. Davis. IRL Press at Oxford University Press, Oxford, 1994
[3] HEK Cell, 24.09.2007 - http://en.wikipedia.org/wiki/HEK_cell
[4] Characteristics of a human cell line transformed by DNA from human adenovirus type 5. Graham FL, Smiley
J, Russell WC, Nairn R. J Gen Virol. 1977 Jul;36(1):59-74.
[5] Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293
cell line. Louis N, Evelegh C, Graham FL. Virology. 1997 Jul 7;233(2):423-9.
[6] LGC Promochem: Cell Biology Collection, 24.09.2007 - http://www.lgcpromochematcc.com/common/catalog/numSearch/numResults.cfm?atccNum=CRL-11268
[7] Tsien R (1998). "The green fluorescent protein". Annu Rev Biochem 67: 509-44.
[8] Green Fluorescent Protein, 24.09.2007 - http://en.wikipedia.org/wiki/Green_fluorescent_protein
41
EXPERIMENT NUMBER: 9
“ANALYSIS OF POLYSACCHARIDES”
9.1. INTRODUCTION
Monosaccharides
A molecule consisting of C, H, and O in a 1:2:1 ratio [(CH2O)n].
Most monosaccharides in protoplasm are either 3-carbon sugars (trioses), 5-carbon
sugars (pentoses) or 6-carbon sugars (hexoses).
The sugars are also characterized by whether they contain an aldehyde (aldoses) or
ketone group (ketoses).
Polysaccharides
Many different kinds of polysaccharides are known, but starch, glycogen and cellulose
are especially important in living systems.
All three are made up entirely of glucose subunits joined by the removal of water
(condensation) to form glycosidic bonds.
They differ strikingly in their structural and chemical properties.
Table 9.1. Properties of three important polysaccharides
Type
Glycogen
Produced by
Animal cells
Starch
Cellulose
Plant cells
Function
Storage polysaccharides that may be
deposited in large granules in cells
Type of glycosidic
bond
α-glycosidic bonds
(glycogen is more highly branched than
starch)
Structural
polysaccharide that
comprises the bulk of
plant cell walls
β-glycosidic bonds
(as are not easily
broken in nature,
cellulose is a very
tough and durable
substance)
Hydrolysis
Breaking of the glycosidic bonds involves the adding back of water across the bond
and thus termed hydrolysis. This can be achieved by heating the polysaccharide in the
42
presence of water and/or strong acids. Enzymes catalyze the same hydrolytic reaction in
normal aqueous solutions without the extreme conditions.
Controls in an Experiment
A negative or a positive control is in a protocol in order to make sure that the result
obtained is not due to;
A protocol that is not capable of producing a positive result (systematic error),
An experimental error in the course of performing the protocol.
In the positive control, a positive effector instead of the tested variant is included, whereas
in a negative control the tested variants are not included and a negative result is expected.
9.2. MATERIALS
1M glucose
toothpicks (a pack)
5M NaOH
0.5% starch
test tubes
Na2CO3
Benedict's solution
Pasteur pipettes
Amylase
6M HCl
9.3. PROCEDURE
9.3.1. Part I
1. Collect as much saliva as possible in the beaker provided.
2. Label 3 tubes as A, B, C, D. Proceed with the experiment as stated in Table 9.2.
Table 9.2
Tube A
15 ml H2O + 30 drops
saliva
Tube B
Tube C
30 drops saliva + 15 ml 15 ml H2O + 30 drops
starch
starch
Incubate at 37° for 1 hr
Add 20 drops Benedict's solution (after 1 hr)
Heat for 5 min in a boiling water bath
Observe and note the color changes
9.3.2. Part II
1. Label 4 tubes as E, F, G, H.
2. Proceed with the experiment as stated in Table 8.3.
Tube D
15 ml starch +
amylase
43
Table 9.3
Tube E
Tube F
Tube G
Tube H
15 ml H2O
15 ml starch soln.
One toothpick broken
into tiny pieces + 15 ml
H2O
15 ml glucose soln.
Heat in a boiling water bath for 15 min and let cool
Add 20 drops of Benedict's solution to all the tubes
Note the colors
Heat in a boiling water bath for 5 min
Note the colors
9.3.3. Part III
1.
Label 3 tubes as I, J, K, L.
2.
Proceed with the experiment as stated in Table 8.4.
Table 9.4
Tube I
Tube J
15 ml H2O + 10 ml 5N
HCl
Tube K
One toothpick broken
15 ml starch solution +
into tiny pieces + 10 ml
10 ml 5N HCl*
5N HCl
Tube L
15 ml glucose soln. +
10 ml 5N HCl
Heat in a boiling water bath for 15 min and let cool
Add Na2CO3 gradually, untill bubble formation ceases
Add 20 drops of Benedict's solution
Heat in a boiling water bath for 5 min
Note the colors
* CAUTION! You should always add acid to water, NOT WATER TO ACID!
** CAUTION! Point the mouth of the tube away from you.
*** CAUTION! Do not add much NaOH, as the reaction proceeds very rapidly.
9.4. QUESTIONS
1. For EACH part of the experiment state;
Negative control tubes
Positive control tubes
Test tubes
2. What does Benedict’s solution contain? Which reaction does it undergo?
44
3. Why did we heat the tubes after adding Benedict’s solution?
4. Why is glucose called a “reducing sugar”?
5. For Part I, what can you conclude about your saliva? What would you expect the
result to be if you had used the toothpick instead of starch solution in the experiment?
6. Please write the reaction that results in bubble formation when Na2CO3 is added in
part. Why do we need this reaction?
7. Draw the structures of,
glucose,
glycosidic bonds in starch and cellulose
the hydrolysis reactions
45
EXPERIMENT NUMBER: 10
“CELLULAR CARBOHYDRATES”
10.1. CELLULAR CARBOHYDRATES
Carbohydrates are a group of macromolecules, which includes simple sugars, and all
larger molecules constructed of sugar subunits. They mainly function as energy store-houses
and as durable building materials. The glycogen in animals and the starch in plants serve as
energy stores. Other carbohydrates, such as chitin and cellulose, have structural roles in
animals and plants, respectively. Oligosaccharides are components of the glycoproteins and
glycolipids found in the plasma membrane. These sugar chains, which always face the cell's
exterior, stabilize the position of glycoproteins and glycolipids within the membrane, function
in cell adhesion, and confer immunological specificity to the cell surface.
Cytology is the study of cells. Every class of macromolecules can be localized in cells
with specific cytochemical reactions. The term cytochemistry can refer to any methodology
that probes the chemical nature of the cell but usually is reserved for specific staining,
reactions and subsequent microscopic analysis. An important cytochemical method used to
identify cellular carbohydrates is the “Periodic Acid-Schiff (PAS) Reaction”. The reaction
stains insoluble polysaccharides (glycogen, starch, cellulose), and the objects of this
experiment are:
to carry out the PAS reaction on fixed blood smears and
to localize PAS-positive material in leukocytes
10.2. HUMAN BLOOD
Human blood is composed of blood cells, cell-like components
and macromlecules suspended in a clear, staw-colored liquid
called plasma. Figure 7.1 shows the components of blood
when it is treated with an anti-coagulant and centrifuged.
Figure 10.1. Components of human blood
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10.2.1. Blood Cells
Blood cells are produced in the bone marrow and arise from a single type of cell called
a pluripotent stem cell.
Figure 10.2. Types of human blood cells
10.2.2. Blood Cell Types
Histologists frequently use Wright-Giemsa stain for the identification and study of
human blood cells. Wright-Giemsa stain is a mixture of methylene blue, methylene azure, and
the eosinates of blood. The azures act as bases and stain the basophilic (base loving) elements
of the cells blue, while the eosins behave as acids and stain the acidophilic (acid loving)
structures red. Since there is a combination of dyes and a varying affinity for each, the various
parts of the cell are stained in hues of pink, purple, blue, and red.
Figure 10.2 is a diagram of the various human blood cell types as they appear in a
fixed smear. There are two classes of cells, red blood cells/erythrocytes and white blood
cells/leukocytes. The leukocytes are placed into two groups, the granulocytes, which have
conspicuous cytoplasmic granules, and the agranulocytes, which lack them. The granulocytes
include the neutrophils, eosinophils, and basophils. The agranulocytes include the
47
lymphocytes and monocytes. In addition to these cell types there are the platelets, which are
small cell fragments.
Erythrocytes: The erythrocyte is the most abundant (about 5 x 10 per µl) and the
smallest blood cell type. Human erythrocytes lack a nucleus and are biconcave. In WrightGiemsa-stain preparations, they appear pink with a central region staining lighter because of
the concavity. They lack any internal organelles but are filled with hemoglobin for the
function of oxygen transport.
Leukocytes: Table 10.1
Table 10.1. Types and properties of human leukocytes
Leukocytes
about 7 x 103/µl leukocytes in human blood
GRANULOCYTES
% among
leukocyte
population
Cell
morphology
AGRANULOCYTES
Neutrophils
Eosinophils
Basophils
Lymphocytes
Monocytes
55-75%
1-5%
0.5%
20-40%
5-7%
Round
Round
Round
Round
Large, round
or oval, some
have blunt
pseudopods
Stain
purple,
may be
round,
indented,
banded, or
lobed
Round and
large, often with
indentation.
They stain dark
purple, and there
are usually
clumps of
chromatin
present
Round or
kidneyshaped, stain
lightly and do
not contain
small, lilacstained
granules
Contain
numerous
large, dark
purple
basophil
granules
Stains blue
-
Production of
antibodies in the
immune
response
Act as
macrophages
in tissues
where they
phagocytize a
variety of
foreign
substances
Morphology
of the
nucleus
when
stained
Dark purple,
multilobed
nucleus
Dark purple
nucleus with two
large lobes
connected by a
thin strand of
chromatin
Morphology
of the
cytoplasm
when
stained
Pale pink
with lightly
stained
granules
Filled with many
large, reddish
orange eosinophil
granules
Function
Body's first
defense
against
invading
microorganisms
Selectively
phagocytize
foreign proteins
that are
complexed with
antibodies
Involved in
allergic
reactions
48
Platelets: Platelets are small, irregularly shaped cell fragments that have broken away
from megakaryocytes in the bone marrow. Platelets are cell fragments produced from
megakaryocytes. Blood normally contains 15-45 x 104 platelets per µl. They range 1-4 µm in
size and stain blue or purple. These numerous cell fragments play a major role in the blood
clotting process.
10.3. PERIODIC ACID-SCHIFF REACTION (PAS)
The periodic acid-Schiff Reaction was first introduced for histological preparations by
J.F.A. McManus in 1946. It is used to stain glycoproteins, polysaccarides, certain
mucopolysaccarides, glycolipids and certain fatty acids in tissue sections.
1. The first step in the PAS reaction is treatment of the fixed cells with periodic acid
(HIO4). HIO4 oxidizes the 2,3-glycol grouping of sugars to a dialdehyde (Figure
10.3). The reaction is very specific, as it forms aldehydes within the polysacchride
molecule but it does not continue the oxidation of the polymers to low molecular
weight water soluble forms.
2. The preparation is then stained with Schiff's reagent, a colorless liquid. Schiff's
reagent reacts with aldehydes to form colored compounds (purplish red). Any cell
structures that stain purplish red with PAS are said to be “PAS-positive”.
3. It is important to note that PAS reaction stains “insoluble” sugars, which contain 2,3glycol grouping.
10.3.2. Slide Preparation
You will be provided with two blood smears that have previously been fixed in a 9:1
mixture of ethanol: formalin. In cytological preparations, cells must be fixed so that their
general morphology, and internal structures will be preserved. If the cells were not treated
with a fixative, the hydrolytic enzymes of the lysosomes would be released and, eventually,
much of the cell would be digested. An effective fixative must render cell components
insoluble, last they be washed out during subsequent treatment. It should also prevent
subsequent swelling or shrinkage of the cell contents. Often, the fixative improves staining by
enhancing the affinity of the cell components for dye molecules. Many fixatives contain an
alcohol plus one or more of the following: acetic acid. formalin, chloroform.
49
For the various steps in the PAS reaction, the fixed blood smears will be transferred to
or stored in Coplin Jars. As the slides are being processed, the slide surface with the cell
preparation must never be allowed to rest against another slide. You will work with two fixed
blood smears, one for the PAS reaction and one for the diastase control. After the control slide
is treated with diastase, both slides are treated with HIO4 followed by Schiff's reagent. The
staining Jar should be kept in the dark since Schiff's reagent deteriorates in the light. After
treatment with Schiffs reagent, the slides are rinsed with a bisulfite bleaching solution.
This rinse assures that the red coloration is due to the cytochemical reaction and not
due to the presence of any basic fuchsin that may be formed during the staining procedure.
Subsequently, the slides are counterstained. Only half of each blood smear will be
counterstained, so that there will be a portion of each smear stained only by the PAS reaction.
The final step in the procedure is the mounting of a coverslip. The coverslip protects the cell
preparation and allows immersion oil to be removed easily.
50
Figure 10.3. The steps in the PAS reaction, shown here a staining portion of a glycogen
molecule
(a)
(b)
(c)
Figure 10.4. The steps for preparation of a thin blood smear
51
10.4. MATERIALS
Coplin jars
200 ml 9:1 ethanol:formalin fixative dispensed in five Coplin Jars (prepare shortly
before use):
180 ml absolute ethanol + 20 ml formalin (i.e., 37% w/w formaldehyde)
300 ml 1% periodic acid solution (good for several weeks if stored in a dark bottle)
dispensed in eight Coplin Jars:
dissolve 3.0g H5IO6 in 300 ml dH2O
100 ml 1N HCl
add 8.1 ml concentrated HCl into 91.9 ml dH2O (in a fume hood)
220 ml Schiff's reagent dispensed in Coplin jars
200 ml Bleaching solution:
180 ml distilled water, 10 ml 10% Na or K metabisulfate solution, 10 ml 1N HCl
100 ml of 10% metabisulfite solution: dissolve 10g Na or K metabisulfite in dH2O
10.5. PROCEDURE
NOTE: All staining procedures are done in Coplin jars; use a zigzag arrangement of up to
nine slides per jar. Always use forceps to place slides in or to remove slides from Coplin Jars.
1. Label the slides with your initials.!!! The markings will also identify the slide surface with
the cell preparation.
2. Smear a tiny drop of blood on the slides. Prepare the blood smear as shown in Figure
10.4. The smear must be very thin and the boundaries should not exceed the cover slip
length. Let the blood on the slides to dry for 8-10 min.
3. Incubate slides in ethanol:formalin fixative for 10 min.
4. Place slides in periodic acid for 10 min.
5. Rinse the slides under running tap water for 1 min (in a Coplin Jar) and then rinse with
distilled water, as in step 3.
6. Place the slides in Schiff's reagent (in a fumed hood) for 10 min. The staining jar should
be kept in the dark.
7. Remove the slides from the staining jar with forceps and transfer them to a clean Coplin
jar. Rinse the slides under running distilled water for several seconds.
52
8. In a fume hood, replace the distilled water with freshly prepared bisulfate bleaching
solution. Decant after 2 min and repeat this bleaching procedure 2 more times.
9. Rinse the slides under running tap water for 5 min and then in distilled water. Wipe the
back of each slide with a tissue paper and allow to air-dry in a vertical position.
10. When the slides are completely dry, place a cover slip and observe using 40X and the
100X objective lens.
10.6. QUESTIONS
1. Why do you think the PAS reaction does not stain nucleic acids?
2. Why do we need to fix cells before further cytochemical techniques?
References:
1. PAS reaction: http://stainsfile.info/StainsFile/stain/schiff/reaction-pas.htm
2. Blood cells background information and Figure 10.2:
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html