BME 4190 Midterm Exam Study Guide
This document is designed to promote critical thinking surrounding the major concepts
presented in each lab. This is not inclusive of the entire course. The exam will not be limited
to this document. It is meant as a study aid for those of you who find a targeted study
approach useful. Answers to this study guide will not be posted or provided; however,
answers can be found in lecture notes and your submitted group reports.
Lab 1 (Cell Processing)
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Three common techniques for cell harvesting are Trypsin, TrypLE, and using a cell
scraper
o How do each work to detach cells ?
o What are advantages and disadvantages of each ?
When using enzymatic methods of harvesting cells we wash cells in PBS, add
Trypsin or TrypLE, incubate at 37 degrees C in the incubator, and then resuspend
cells in media to stop dissociation.
o Why do we rinse in PBS instead of media ? (what components does PBS not
have)
o Why do we put the flask in the incubator instead of leaving it at room
temperature ?
o How does adding media inactivate the dissociation agent ?
We culture cells in media with a phenol red pH indicator. Why?
How is a hemocytometer used to count cells?
How can you calculate cell concentration and total number of cells from knowing
the counts from a hemocytometer?
Why did we use Trypan Blue and how does it work?
Why is there DMSO in freezing media ?
We used a Mr. Frosty – a container containing alcohol – to freeze cells , what did this
do?
Methods to analyze cell growth include cell counting, a DNA assay, and an MTT
assay. (see exercise question in lab A report)
Exercise questions from lab report A
1. Using the cell counts that you determined following cell dissociation with ‘trypsin’ and
reported in Table 2, how much of your cell suspension would you need to seed into each
well of a 24-well plate for an experiment that requires an initial seeding density of 5,000
viable cells per cm2? Note that a 24 well plate = 2 cm2
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Number of cells required per well = 5000 cells/ cm2 * 2 cm2 = 10,000 cells per well
Assume the wells have media in them already so we only need to find the volume of
the original cell suspension that contains 10,000 cells that we would add to the well
For an example let’s say the average number of viable cells (white, excluding trypan
blue) is 50 cells per quadrant of the hemocytometer , and the average number of
non-viable cells (blue, took up the trypan blue die) per quadrant is 2.
To calculate the viable concentration of the cell suspension we use the following
equation
o Average number of viable cells/quad * 10,000 * 2 = concentration of cells in
the suspension in units of cells/mL
▪ The 2 in the equation there because we diluted cells 1:1 in trypan blue
o 50 cells/ quad * 10,000 *2 = 1,000,000 cells per mL
We need to find the volume of the cell suspension that will give us 10,000 cells to
add to each well
o (unknown volume) * 1,000,000 cells/mL = 10,000 cells
o 10,000 cells / 1,000,000 cells /mL = 0.01mL = 10uL of cell suspension
o We would add 10ul of this suspension to each well to get a seeding density of
5000 cells/ cm2
2. Briefly describe two alternative methods that could have been used to determine the
number of cells and cell viability in your experiments. Cite your references.
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DNA assay (cell number)
MTT assay (number of viable cells)
ATP assay (number of viable cells)
Live/dead staining with PI and Calcein AM (number of viable cells)
Answers may vary - please see the following paper for additional details
o https://www.ncbi.nlm.nih.gov/books/NBK144065/
Lab 2 (Cell Cycle analysis via Flow Cytometry)
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How does DNA content change in each phase of the cell cycle ?
What does PI stain? What has to be done to the cell to get the dye in?
How would activation of a cell cycle checkpoint impact PI staining intensity in flow
cytometry results (how would the peaks be shifted) ?
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In class we talked about thymidine, BrdU, or Edu incorportation. These stains work
when the dye is added to live cells and incorporated into DNA that is actively being
synthesized. What advantages does PI staining have over these methods?
Flow cytometry uses visible light scattering to determine basic parameters of cells
used for gating.
o What does side scatter tell you about a cell?
o What does forward scatter tell you about a cell?
o We drew gates in lab – what was each gate trying to include or exclude ?
Why did we have an unstained control and a stained control in the PI lab?
Why do we treat cells with RNAse prior to flow cytometry ?
Exercise questions from lab report B
1. MSCs can grow in vitro for 10 to 20 passages, but, like all other primary cells, are
subject to the Hayflick limit. Therefore, human MSCs may become senescent
following extended culture. What does senescence refer to and how does it affect
the differentiation, telomere length, and morphology of MSCs? Cite your references.
Please see here:
• https://www.nature.com/articles/35036093 PMID: 11413492
• https://www.frontiersin.org/journals/cell-and-developmentalbiology/articles/10.3389/fcell.2021.645593/full
• Binato R, et al.(2013) Stability of human mesenchymal stem cells during in
vitro culture: considerations for cell therapy. Cell Prolif46(1):10-22.
https://www.ncbi.nlm.nih.gov/pubmed/23163975
2. How would you expect your cell cycle analysis to change with increasing passage
number (before reaching the Hayflick limit)? How do you expect the respective
phases of the cell cycle to change in senescent MSCs (after reaching the Hayflick
limit) relative to non-senescent MSCs? Explain your reasoning and cite your
references.
a. Answer not provided
b. please consider what phase cells are in when the Hayflick limit is reached
and what phases they will not enter anymore because they will not divide any
more
c. Please consider the three phases of the cell culture described by Hayflick
Lab 3 (Differentiation and cell staining)
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We differentiated MSCs down adipogenic and osteogenic pathways
o What does Oil Red O stain for and what differentiation condition did we
expect to stain positively ?
o What does Alizarin Red stain for and what differentiation condition did we
expect to stain positively?
MSC can self-renew as stem cells or differentiate down a variety of linages. Cell
density is proportional to differentiation capacity. How would you alter cell culture
conditions to promote self-renewal?
What challenges did the high density used here pose for image analysis?
Exercise questions from lab report D
1. Which other tests (i.e., instead of Oil Red O staining or qPCR) could you run to confirm
adipogenic differentiation at experimental endpoints? Name and describe 2 alternative
assays.
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Hint: you do not need to limit your search to adipogenic differentiation of MSCs , the
fibroblast cell like 3T3-L1 area also commonly differentiated into adipocytes so you
can check papers that reference them
o Are there alternative lipid stains?
o Are there adipogenic lineage-specific markers you can stain for using
antibodies ?
o Other methods of quantifying gene expression?
o Anything that these differentiated cells secrete that could be collected from
the media and analyzed ?
2. Which other tests (i.e., instead of Alizarin Red staining or qPCR) could you run to confirm
osteogenic differentiation at experimental endpoints? Name and describe 2 alternative
assays.
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Please reference the literature
Hint: we looked at ALP gene expression but is there a way to functionally investigate
if ALP is active and present in a culture?
Lab 4 (RNA isolation)
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Why did we isolate RNA instead of DNA? What does RNA tell us about the
differentiation state of the cells that DNA would not?
Next, we performed a reverse transcription reaction to convert the RNA to cDNA.
What is the major advantage of cDNA ?
Why did we need to use the RNA easy spin columns ? What happened to all of the
other cell components that we added to the columns?
Why did we use the spectrophotomter before reverse transcription? What did the
260/80 ratio tell us ?
Lab 5 qPCR
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PCR stands for Polymerase Chain Reaction- why is it called a chain reaction ?
To run PCR you have to identify a target gene of interest and design a primer set that
will specifically amplify that sequence of interest
o In class we talked about how to use primer blast to design a primer set
▪ When you get a list of results of potential primers what are 2 examples
of criteria you would use to choose one primer set over another ? In
other words what are 2 of the criteria you would use when designing a
primer set
Why did we have a no template control for each primer set?
What is a key criteria to use when choosing a reference/housekeeping gene? Why do
we need a housekeeping gene?
qPCR works by quantifying the amount of fluorescently labeled double stranded
DNA after each cycle. The Ct value is the number of cycles it took for the
fluorescence intensity to rise above a threshold value. What is the relationship
between Ct number and concentration of the target gene in the cDNA present in the
sample? In other words, does a high Ct value mean the gene is highly or lowly
expressed in the sample?
What information can be gained from a melt curve plot and a negative first derivative
plot? An ideal first derivative plot would have only one large peak signifying the
target amplified region of cDNA. What would a smaller peak at a lower temperature
signify? A larger second peak at a high temperature ?
The ΔΔCT Method was used to assess relative gene expression. What does this
mean?
Exercise Questions from lab report C
1. We used the RNAeasy kit to isolate RNA from your lysed samples. Is the final eluted RNA
just mRNA or are there other constituents in there? What was the purpose of the DNase
step?
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See lecture and the resources on the Qiagen website for the RNAeasy kit
https://www.qiagen.com/us/products/discovery-and-translational-research/dnarna-purification/rna-purification/total-rna/rneasy-kits
2. In this lab, we used ΔΔCT Method to assess relative gene expression of markers of
adipogenic and osteogenic differentiation. If you are just comparing your experimental
condition to your control condition, what is the point of using the housekeeping gene per
sample and how would you need to modify your analysis to determine absolute changes in
gene expression?
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https://www.qiagen.com/us/resources/faq?id=7aaa70e5-73df-4533-a8eeae180a88cd08&lang=en
The qPCR readout is a fluorescence intensity (from labeled double stranded DNA)consider what that value means if you had no other information besides the intensity
from a gene of interest. How would we use the arbitrary fluorescence value? What
other information would be need to turn the fluorescence value into a number that
relates to gene expression in the sample?
3. Both PPARγ and ALP are relatively early markers of adipogenesis and osteogenesis,
respectively. Please identify one late marker of differentiation for each adipogenesis and
osteogenesis, design primer sequences for these markers, and provide a brief description
of how you would modify your experiment to measure and validate these.
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Please see Week 8 lecture