BASIC EXERCISES
FOR THE INTRODUCTION TO
ANIMAL CELL CULTURE
CONTENTS
1A. Practicing Sterile Technique in a Vertical Laminar Flow Hood
1B. Thawing a Cryovial of NIH3T3 Cells to Initiate Cell Culture
1C. Use of Trypsin for splitting a culture of NIH3T3 cells
1D. Using the Hemocytometer
Protocols developed by
Robert M. Nissen and Sandra Sharp
Department of Biological Sciences
California State University, Los Angeles
page 2
page 6
page 9
page 13
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1A: Practicing Sterile Technique in a Vertical Laminar Flow Hood
As you proceed through this set of protocols and experiments, be sure to enter all your work into your
notebook!
Objectives
Introduction to sterile pipetting in a laminar flow hood
Improve dexterity with sterile pipetting in a laminar flow hood
Learn and adhere to safety considerations
Vocabulary
Vertical laminar flow, horizontal laminar flow, medium, media, sterile
Outline
Obtain practice solutions and vessels; transfer liquid volumes to make media without spills; transfer and
manipulate plates without spills.
Materials
sterile
0.5% Phenol red
TE, pH 8 (10mM Tris, 1mM EDTA)
175mM acetic acid (1:100 dilution from glacial acetic acid)
serological pipettes
plastic pipette tips (barrier)
50 ml conical screw cap tubes
non-sterile
37C water bath, maintained with antimicrobial additive
rack in water bath that will securely hold vials with cap above water level
rack in laminar flow hood that will securely hold cryovial
fully-stocked biosafety cabinet
kimwipes for swabbing
70% ethanol
DAY 1 (Monday)
Protocol
PREPARATION
1. Prepare the hood
a. The hood should initially be found with the sash pulled down. If it is not, close the sash.
b. If the hood is equipped with UV, you can turn on the UV with the sash closed for 30
minutes prior to initiating work in the hood.
i. UV light poses significant health risks to the investigator and cannot sterilize
crevasses or items shielded from the rays. Thus, UV sterilization should not be
used as the sole method for hood sterilization and can be completely replaced
by alternative methods.
c. Raise the sash and inspect the hood to ensure airflow is on at an acceptable flow rate.
i. Minimum flow rates to maintain laminar flow are 0.4m/s (80ft/min).
d. Clutter within the hood should be kept to a minimum and any materials should be
positioned within the hood to minimize disruption of the laminar airflow.
i. Never block the front or rear vents.
e. Using large kimwipes, swab down the work surfaces with 70% ethanol and wipe clean.
3
2. Collect all materials
a. Place only the required sterile items within the hood necessary for the task at hand.
b. All materials entering the hood should be sprayed and/or wiped down with 70% ethanol
to sterilize their outer surfaces.
i. Remember, your hands are on the list of items that must be sterilized every time
they exit the hood before reentry.
ii. When working with BSL 1 rated materials (such as today), gloves are not
required.
c. For today, items to be sterilized include:
i. Containers of the various stock solutions (phenol red, etc)
ii. Aspirator and corresponding pipettes
iii. Plastic pipette tip boxes and pipettors (as necessary based on your calculations)
iv. Two 10 cm plates, two 6 cm plates, a 15 ml conical screw cap tube, a 50mL
conical screw cap tube
v. Appropriate sized tube racks
LABEL THE VESSELS
3. Label all containers prior to beginning any transfers of liquids between containers.
a. After reading through the entire protocol, prelabel the plates. Include today’s date,
your initials, and your group number as well as any additional details that might be
needed in order to tell your plates apart.
b. Prelabel any sterile screw cap tubes to be used.
PREPARE THE MEDIUM
4. General guidelines:
a. Read through all steps before beginning.
b. Loosen the screw caps for the various (thawed) components, but do not remove them
from the various vessels. Be careful not to put your hands at the point where the cap
and the container meet. This is a vulnerable location for contamination.
c. Using a fresh and sterile pipette for each different item, mix each solution by gentle
swirling or by pipetting up and down a few times. Do not mix by inversion. Avoid
getting liquid up into the neck of any container.
d. Take special care to not touch the pipette tip to any unintended surfaces. Likewise, do
not touch insides of caps, edges or rims of containers with your hands.
e. If dexterity allows, do not set vessel lids down on any surface at any time. Hold the cap
until the desired volume has been removed and then immediately replace the cap on
the vessel. If you cannot manage this, place the cap open side DOWN on the sterilized
work surface of the hood. Do not rely on your partner. It is important to learn to carry
out these manipulations on your own.
f. A good rule of thumb is to pipette the largest volume first and smallest volume last.
g. After each component has been added, tighten the screw cap on the corresponding
reagent vessel.
h. After all components have been added, ensure the stock bottles are securely tightened
and then remove them from the workspace to reduce clutter.
i. Ensure the solution has been mixed to homogeneity by gently swirling the capped vessel
within the hood.
5. Aliquot 39.6ml of TE, pH 8 to a 50mL conical tube.
6. Add 0.4ml of 0.5% phenol red stock and mix gently by pipetting up and down. This is your
“medium” for these exercises. It should look red.
4
ADD MEDIUM TO PLATES
7. Medium plates:
a. Transfer 10mL of medium to each of two 10 cm plates and 5mL to the two 6 cm plates.
i. Hold the plate lid by the edges (the angle between the top and the sides) above
the plate just enough for the pipette to dispense media into the plate.
ii. Be sure to dispense the solution from the pipette gently so as not to create any
splash. Harsh pipetting can also dislodge some cell types from the bottom of the
dish.
8. Distribute the medium evenly by very gently rocking the plate.
a. Be sure not to swirl the plate as this tends to concentrate cells at the edges. Rock or
slide the plate from side to side a few times. Allow the medium to come to a rest, and
then rock or slide the plate from front to back. Take special care not to spill the liquid or
to get the lids of the plates wet.
b. Transfer the plate stacks to the incubator without spilling any contents or getting the
lids of the plates wet. If you spill or get any medium on any part of the lid, start over in
order to improve your technique.
9. Acidification of medium plates:
a. Retrieve the 10cm and 6cm plates from the incubator.
b. Add 150uL of 175mM acetic acid to one of the 10cm plates and 75uL to one of the 6cm
plates. Mix by gently rocking the plates as described above
i. Take special care not to spill the liquid or to get the lids of the plates wet.
ii. If solutions were made correctly, the solution should appear slightly orange.
This is what plates look like when cells need fresh medium (or might be
overgrown).
iii. For perspective, domestic vinegar is typically between 0.5-1M acetic acid.
c. Add an additional 75uL to the 6cm plate. Mix by gently rocking the plate.
i. The solution should appear quite yellow. These would be plates of mostly dead
cells. Lets all hope that our real plates never look like this.
10. Feeding your cells:
a. Use the aspirator to remove the liquid from the acidified plates.
i. Aspirate medium at one edge of the plate, moving the tip from the surface of
the liquid down the inner wall of the plate until almost all liquid is removed.
ii. Tipping the plate slightly will facilitate aspiration of the liquid medium.
iii. Do not touch the aspirator tip to the floor of your plates. This is where your cells
would be and doing so can pull them off the plate and into the aspirator.
iv. Do not allow the “cells” on your plates to sit without medium for more than a
couple minutes.
b. Add fresh medium to the 6cm plate dribbling the medium into the plate from an edge.
i. Do not blast the media into the center of the plate as this can dislodge cells
from the plate.
11. Return your plates (one 10cm plate and two 6cm plates) to the 37C incubator.
12. Tightly cap and store your remaining medium in the 50mL conical tube. Since this is not real
medium, it can be stored at room temperature.
DAY 2 (Wednesday)
Protocol
1. Remove your plates from the incubator and inspect the color of the medium in the plates.
Compare it to the medium you stored in the 50mL conical tube.
2. When finished, aspirate and properly dispose of the materials.
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Questions
1. Phenol red is a pH indicator dye that is purple at alkaline pH, red near neutrality, orange at
slightly acidic pH and yellow at acidic pH.
a. Why would medium exposed to growing cells gradually turn yellow?
b. What effect would you expect from exposing your medium to a 5% CO2 environment?
2. Why is it bad to leave your cells without medium for prolonged periods of time while in the
hood?
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1B: Thawing a Cryovial of NIH3T3 Cells to Initiate Cell Culture
As you proceed through this set of protocols and experiments, be sure to enter all your work into your
notebook!
Objectives
Improve dexterity with sterile pipetting in a laminar flow hood
Become familiar with thawing and plating procedures
Learn and adhere to safety considerations
Vocabulary
cryovial, medium/media, cell culture, confluence
Outline
Obtain a cryovial containing NIH3T3 cells; thaw cells quickly; dilute them, and reseed them at high cell
density.
Materials
sterile
one cryovial containing NIH3T3 cells/team of 2
one 10 cm plate/team of 2
3T3 growth medium components
serological pipettes
plastic pipette tips (barrier)
15 ml conical screw cap tubes
50 ml conical screw cap tubes
non-sterile
37C water bath, maintained with antimicrobial additive
rack in water bath that will securely hold vials with cap above water level.
rack in laminar flow hood that will securely hold cryovial
fully-stocked biosafety cabinet
kimwipes for swabbing
70% ethanol
DAY 1 (Wednesday)
Protocol
PREPARATION
13. Prepare the hood (UV, airflow, vents, 70% ethanol)
14. Collect all materials
a. For today, items to be sterilized include:
i. Components for preparing the 3T3 growth medium (see your media tables)
ii. Serological pipettes and pipette aid
iii. Aspirator and corresponding pipettes
iv. Plastic pipette tip boxes and pipettors (as necessary based on your calculations)
v. A 10 cm plate and a 15 ml conical screw cap tube per team of two.
vi. One or two 50 ml conical screw cap tubes
vii. Appropriate sized tube racks
15. Prepare the medium.
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a. Loosen the screw caps for the various (thawed) components. Be careful not to put
your hands at the point where the cap and the container meet. This is a vulnerable
location for contamination.
b. Read through all parts of this step before beginning. Using a fresh and sterile pipette for
each different item, mix each solution by gentle swirling or by pipetting up and down a
few times. Do not mix by inversion. Avoid getting liquid up into the neck of any
container. Pipette the appropriate volume into the vessel for the 3T3 growth medium.
i. Consult the media table to determine the various required volumes. You will be
using the vessel containing the DMEM as the growth medium vessel and will
therefore need to remove some of the DMEM from that container. Save the
excess DMEM in labeled sterile 50mL conical tubes for later use.
ii. Take special care to not touch the pipette tip to any unintended surfaces.
Likewise, do not touch insides of caps, edges or rims of containers with your
hands.
iii. Loosen caps, but do not remove them from the various vessels.
iv. If dexterity allows, do not set vessel lids down on any surface at any time. Hold
the cap until the desired volume has been removed and then immediately
replace the cap on the vessel. If you cannot manage this, place the cap open
side DOWN on the sterilized work surface of the hood. Do not rely on your
partner. It is important to learn to carry out these manipulations on your own.
v. A good rule of thumb is to pipette the largest volume first and smallest volume
last.
vi. After each component has been added, tighten the screw cap on the
corresponding reagent vessel.
vii. After all components have been added, ensure the stock bottles are securely
tightened and then remove them from the workspace to reduce clutter.
viii. Ensure the 3T3 growth medium has been mixed to homogeneity by gently
swirling the capped vessel within the hood.
ix. If the 3T3 growth medium is not warm, prewarm it in the 37C waterbath for 5
minutes.
16. Label the culture plates.
a. Prelabel the 10 cm plate. Remember to label in small printing near the edge of the
cover. The label should include the name of the cell line (NIH3T3), the date on which the
cells are thawed (which happens to be today), today’s date, your initials, and your group
number.
i. Example: NIH3T3, 9/29/11-9/29/11, RMN, Group 42
ii. Please note, in future sessions, the thawed date will change only if working from
a new vial of thawed cells. Thus you will always be able to look at your plates
and know how long the cells have been in culture and when they were last split.
b. Prelabel a sterile screw cap 15 ml tube.
17. Pipette 10 ml prewarmed 3T3 growth medium into the 10 cm plate.
THAWING & PLATING
18. Retrieve one ampoule of cells, checking the label to be sure you have the correct cells.
19. As quickly as possible, transfer the vials to a rack in a 37C water bath that is maintained with
antimicrobial additive.
a. Avoid getting water up to the cap, as this will increase the chances of contamination.
b. If there is no rack, hold the tube near the cap without letting your fingers cross the gap
between lid and vial, and allow the lower half of the vessel to enter the water. Wait and
hold.
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20.
21.
22.
23.
24.
25.
c. When the contents have thawed, swab the ampoule thoroughly with 70% alcohol, and
loosen the lid in a laminar flow hood.
Gently pipette the ampoule contents up and down twice and transfer the contents to the
correspondingly labeled 15 ml tube.
Very slowly, drop by drop at first, and then more quickly, add an additional 4.5 mls prewarmed
3T3 growth medium to the 15 ml tube and mix by pipetting up and down gently.
Spin the capped tube at 100g for 10 minutes in a clinical table top centrifuge. Aspirate off the
medium, being careful not to disturb the pellet of cells.
a. It is acceptable to leave 0.5 ml of media at the bottom of the tube to ensure that you
have not disturbed the cell pellet.
b. If you aspirate your cells, they are gone forever since the aspirator contains bleach that
will instantly kill them.
Gently resuspend the pellet in 5 mls prewarmed medium and transfer the entire cell suspension
to the correspondingly labeled 10cm plate. Distribute evenly by very gently rocking the plate.
Be sure not to swirl the plate as this tends to concentrate cells at the edges. Rock or slide the
plate from side to side a few times. Allow the medium to come to a rest, and then rock or slide
the plate from front to back.
a. Take very particular care not to spill any medium from the plate or to even get the lid
wet with medium. Doing so will greatly increase the likelihood of later contamination. If
you do get medium on the lid, aspirate it off with a sterile Pasteur pipette, remembering
not to turn the lid under side up.
Clean any spills with ethanol.
Place the plate in the 37C incubator in the section reserved for your team.
DAY 2 (Friday)
Protocol
1. Confirm attachment. Observe your cells under the microscope and estimate the % confluence.
(See protocol 1.3)
2. Refeed the plate of cells or trypsinize and replate them if the cells are likely to reach >70%
confluence by Monday. (See protocol 1.3)
a. You will need these cells for subsequent experiments.
b. Remember to refeed/passage your cells each lab session.
Questions
1. If you wanted to predict the % confluence you would likely observe on Friday, what information
would you need?
2. Why might spilling medium out of a plate increase the chance of a contaminating
microorganism entering the plate?
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1C: Use of Trypsin for splitting a culture of NIH3T3 cells
As you proceed through this set of protocols and experiments, be sure to enter all your work into your
notebook!
Objectives
Improve dexterity with sterile pipetting in a laminar flow hood
Become familiar with feeding and splitting procedures for adherent cell cultures
Learn and adhere to safety considerations
Vocabulary
trypsinization, PBS
Outline
Aspirate old medium; wash cell monolayer with PBS; add trypsin to cells; uniformly disperse cells by
repeated pipetting; inactivate trypsin by addition of serum-containing medium; distribute cells to
multiple plates containing fresh medium.
Materials
sterile
trypsin solution
phosphate buffered saline (PBS), at room temp
3T3 growth medium mix (from previous session), prewarmed to 37C
two to four 10 cm plates/team of 2
serological pipettes
plastic pipette tips
non-sterile
37C water bath, maintained with antimicrobial additive
rack in water bath, as necessary
fully-stocked biosafety cabinet
kimwipes for swabbing
70% ethanol
DAY 1 (Friday)
Protocol
PREPARATION
26. Prepare the microscope and assess confluency
a. Remove the protective plastic cover from the microscope
b. Sterilize the microscope stage area by spraying 70% ethanol on a kimwipe and wiping
the stage area and knobs.
i. Do not spray the microscope as this may soil the objective lenses and filter sets
c. Turn on the inverted microscope light source
d. Remove a cell culture plate from the incubator and place it on the microscope stage
e. Use the microscope to assess the confluency of the cells.
i. 100% confluency means that all surface area is occupied by cells
ii. 50% confluency means half of the surface area is occupied by cells, and so on.
f. Use the digital camera to record an image of your cells.
g. Depending on the cell type and application, determine whether the cells need to be fed
or split.
10
27.
28.
29.
30.
i. Allowing cultures to reach high confluence (80-100%) might permanently
alter some cell behaviors and, therefore, is to be avoided. Always seek specific
guidance when working with a new cell line/type.
ii. For NIH3T3 cells, at 70% confluency, they should be split to new plates.
h. Return the plate of cells to the incubator while preparing the hood.
i. If done with the microscope, turn off the light and replace the protective covering.
Prepare the hood
Collect all materials
a. For today, items to be sterilized include:
i. Trypsin bottle
ii. PBS bottle
iii. 3T3 growth medium bottle, prewarmed
iv. Serological pipettes and pipette aid
v. Aspirator and corresponding pipettes
vi. Plastic pipette tip boxes and pipettors (as necessary based on your calculations)
vii. 10 cm plates
Label the appropriate number of new culture plates.
a. Remember to label in small printing near the edge of the cover. The label should include
the name of the cell line (NIH3T3), the date on which the cells are thawed, today’s date,
your initials, and your group number.
i. Example: NIH3T3, 9/28/11-9/30/11, RMN, Group 42
ii. The number of plates you split into depends on the starting confluency,
doubling time for the cell line, and number of days before the next split.
Loosen the screw caps for the various (thawed) components
WASHING ADHERENT CELLS WITH PBS
31. Retrieve the cell culture plates from the incubator
32. Aspirate the old medium from the plates
a. Aspirate medium at one edge of the plate, moving the tip from the surface of the liquid
down the inner wall of the plate until almost all liquid is removed.
i. Tipping the plate slightly will facilitate aspiration of the liquid medium.
ii. Do not touch the aspirator tip to the floor of your plates. This is where your cells
are and doing so can pull them off the plate and into the aspirator.
iii. Do not allow the cells on your plates to sit without any aqueous solution on
them for more than a couple minutes.
33. Add 5ml of PBS to each aspirated cell culture plate
a. Take special care to not touch the pipette tip to any unintended surfaces. Likewise, do
not touch insides of caps, edges or rims of containers with your hands.
b. If dexterity allows, do not set vessel lids down on any surface at any time. Hold the cap
until the desired volume has been removed and then immediately replace the cap on
the vessel.
c. Hold the plate lid by the edges above the plate just enough for the pipette to dispense
media into the plate.
d. Be sure to dispense the solution from the pipette gently so as not to create any splash
or dislodge the cells.
e. Disperse the medium evenly by gently rocking the plate(s).
f. Take special care not to spill the liquid or to get the lids of the plates wet.
g. 20 seconds of gentle washing is sufficient
11
TRYPSINIZATION OF ADHERENT CELLS
34. Aspirate the PBS from the plates
35. Add 1ml of prewarmed Trypsin solution to each 10cm plate. Scale the volume downward
accordingly for plates with smaller amounts of surface area. (For now, we will leave the solution
on the cells to avoid dessication. As your efficiency improves it will be possible to remove the
trypsin solution.)
a. With the lid replaced on the dish, gently rock the plate(s) to ensure complete coating of
all cells on the plate with the trypsin solution.
b. The length of time needed to release cells from the plate varies for different types of
cells. For NIH3T3 cells, 2 to 3 minutes should be sufficient. Check by periodically tilting
the plate and looking for the cell monolayer to “slip” from the plate surface. If you are
unsure, you can check on the microscope. The cells will become spherical as they
detach from the plate and ECM.
c. Gently tapping the upright plate against an object, such as the side of the tissue culture
hood wall or the floor of the hood, can facilitate the “slipping” of the cell monolayer
from the plate surface. YOU DO NOT NEED TO CONSTANTLY DO THIS TO YOUR CELLS.
36. While waiting for the trypsinization to occur, pipette 10 ml prewarmed 3T3 growth medium into
the desired number of fresh 10 cm plates.
37. Once the cell monolayer is mostly released from the plate surface, add 2 to 4mL of growth
medium to the plate.
a. The serum in the growth medium contains protease inhibitors that will block further
activity of the trypsin enzyme.
b. If splitting more than one plate of cells, the cells and solution from one plate can be
combined with that from additional plates to yield a “cell stock”.
38. Once all the desired cells are collected, suck them up into the pipette, place the pipette opening
firmly against the inside growth surface of the old plate, and completely dispense the liquid back
into it to mechanically disperse the cells. Repeat this process 3-4 times.
Note: Consider the volume you are working with! 15mLs of solution cannot be
completely sucked up into a 10mL pipette without damaging the protective filter within
it!
REPLATING TRYPSINIZED CELLS
39. Pipette the desired volume of cells into each fresh plate containing prewarmed 3T3 growth
medium to seed the cells at the desired initial confluency.
a. It is common to not replate all cells if there are more cells than needed for a particular
downstream application.
b. If not all the cells are to be replated, once the desired amount are replated, dispose of
the remainder by aspiration.
Note: Due to gravity, cells in suspension will settle out of solution, concentrating at the
bottom of the container. Resuspend the “cell stock” by swirling between each pipetting.
40. Gently rock the fresh plates to evenly distribute the cells.
a. The inverted microscope can be used to assess the effectiveness of the redistribution of
the cells. A common problem is for cells to clump into the center or outer edges of the
plate if you “swirl” the plates.
41. Place the fresh plate(s) in the 37C incubator.
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DAY 2 (Monday)
Protocol
3. Confirm attachment. Observe your cells under the microscope and estimate the % confluence.
4. Refeed or split if the cells are likely to reach >70% confluence before the next lab session.
Table of cell culture growth areas for various sizes of dishes and plates.
Dish or
plate size
15cm
10cm
6cm
3.5cm
6-well
12-well
24-well
48-well
96-well
384-well
Diameter
(cm)
15
10
6
3.5
3.5
2.2
1.6
1.1
0.64
0.27
Growth Area
(cm2)
176
78
28
9.6
9.6
3.8
1.9
0.95
0.32
0.056
Working
Volume (mL)
20-40
10 - 16
4-6
2.0 - 3.0
2.0 - 3.0
0.75 - 1.25
0.40 - 0.60
0.20 - 0.30
0.10 - 0.20
0.025 - 0.050
*note: these are approximate values that vary from one manufacturer to another.
Questions
3. Starting with a 10cm plate of cells at 80% confluency on a friday, you split the cells from that
plate to four fresh 10cm plates.
a. What is the predicted initial confluency of the cells on the fresh plates?
b. Assuming a doubling time of 24 hours, when will the cells again require splitting?
c. Do you look forward to tending your cells on Sundays?
4. Starting with a 10cm plate of cells at 75% confluency, propose a splitting scheme that will yield
plates approaching 70% (+/- 5%) confluency between weekdays and a separate splitting plan for
across the weekend.
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1D: Using the Hemocytometer
As you proceed through this set of protocols and experiments, be sure to enter all your work into your
notebook!
Objectives
Improve familiarity with the hemocytometer
Vocabulary
adherent, hemocytometer
Outline
Trypsinize and count cells using the hemocytometer; distribute cells at specified densities to plates of
different sizes; determine the doubling time for your cells.
Materials
sterile
PBS, trypsin, 3T3 growth medium
two 10cm plates
six 6cm plates
serological pipettes
plastic pipette tips
non-sterile
37C water bath, maintained with antimicrobial additive
Trypan blue solution
hemocytometer
rack in water bath, as necessary
fully-stocked biosafety cabinet
kimwipes for swabbing
70% ethanol
DAY 1 (Monday)
Protocol
PREPARATION
42. Prepare the microscope and assess confluency
43. Prepare the hood
44. Collect all materials
a. For today, items include:
i. Trypsin, PBS and 3T3 growth medium bottles
ii. Required tools (pipettes, pipetting devices, aspirator)
iii. Two 10 cm plates and a six 6cm plates.
45. Label the new culture plates.
a. In addition to the usual labeling, also label the plates with the quantity of cells to be
seeded (see later in the protocol).
46. Loosen the screw caps for the various (thawed) components
WASH AND TRYPSINIZE ADHERENT CELLS
47. Retrieve the cell culture plates from the incubator
48. Aspirate the old media from the plates
49. Add 5ml of prewarmed PBS to each aspirated cell culture plate
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50. Aspirate the PBS from the plates
51. Add 1ml of prewarmed Trypsin solution to each 10cm plate. Scale the volume downward
accordingly for plates with smaller amounts of surface area.
52. While waiting for the trypsinization to occur, pipette 10 ml prewarmed 3T3 growth medium into
the desired number of fresh 10 cm plates and 5 ml into each 6 cm plate.
53. Once the cell monolayer is mostly released from the plate surface, add 4mL of growth medium
to the plate.
54. Once all the desired cells are collected, mechanically disperse the cells by repeated pipetting.
a. To obtain consistent results from one day to the next, settle into a style that works for
you and stick to it every time you split cells (for example, always dispersing the cells 3
times versus 2, etc).
b. This is your cell suspension stock.
Figure 1. The hemocytometer
Figure 2. Neubauer Improved grid (detection area)
USING THE HEMOCYTOMETER
55. Obtain a clean hemocytometer. Be careful, the glass is fragile and they are expensive!
56. Into an eppendorf tube, combine 20uL of cell suspension with 20uL of Trypan blue solution.
57. Load 10uL of the mix onto the sample area (see Figure 1.)
a. Be careful to avoid introducing any bubbles.
b. The grid is fragile, do not scratch it.
c. Add coverslip across the ‘bridge’.
58. View the appropriate detection area on the microscope and count the number of cells that are
not blue in the 4 large corner squares (see Figure 2, each large corner square is composed of 16
small squares).
**DO NOT LET THE CELLS DRY ONTO THE HEMOCYTOMETER**
**VERY GENTLY WIPE THE SURFACE CLEAN WITH A KIMWIPE**
a. Using the equation above, calculate the concentration of live cells in your undiluted cell
suspension stock.
b. The (2) in the above equation corrects for the dilution of the sample with Trypan blue.
59. If the cells in the detection area are too dense to yield an accurate count, return to the hood
and create a small amount of an appropriate dilution of the original cell solution.
a. For example, you may wish to try a 1:10 dilution by adding 100uL of cells from your
stock to 900uL of PBS. Then, combine 20uL of the diluted cell suspension with 20uL of
Trypan blue solution and load the hemocytometer again.
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b. When repeating the calculation remember to include the appropriate multiplier for
your additional dilution factor.
REPLATING TRYPSINIZED CELLS AT SPECIFIED CELL DENSITIES
60. Based on the concentration of your cell stock, determine the volumes to be added to the various
plates and wells in order to achieve the seed numbers below:
a. 10cm plate at:
(a 1:4 split)
b. 10cm plate at:
2x105 cells
c. 6cm plate at:
2x105 cells
d. 6cm plate at:
105 cells
e. 6cm plate at:
5x104 cells
f.
2.5x104 cells
6cm plate at:
g. 6cm plate at:
104 cells
h. 6cm plate at:
5x103 cells
61. Pipette the calculated volume of cells into each fresh plate containing enough prewarmed 3T3
growth medium to yield a final volume of 10mL for each 10cm plate or 3mL for each well of the
6-well plate.
a. Remember gravity is acting on your cell stock! Swirl to resuspend!
b. If not all the cells are to be replated, dispose of the remainder by aspiration.
62. Gently rock the fresh plates to evenly distribute the cells.
63. Place the fresh plate(s) in the 37C incubator.
DAY 2 (Wednesday)
Protocol
5. Confirm attachment. Observe your cells under the microscope and estimate the % confluence
for each seeding condition.
6. From the 6-well plates, choose one that is between 25% and 50% confluency, trypsinize the cells
from the well and use the hemocytometer to calculate the total number of live cells in the
well.
7. Refeed or split one of the 10cm plates if the cells are likely to reach >70% confluence before the
next lab session (See next protocol).
Questions
5. Based on your methods and calculations, how many NIH3T3 cells are on:
a. a 100% confluent 10cm plate?
b. a 100% confluent 6cm plate?
6. Based on your calculated total number of live cells in one of the 6cm plates, what is the
approximate doubling time for NIH3T3 cells?
Note: given an initial total cell number (c1), a growth time of (h) hours, and a final total
cell number (c2), the doubling time (D) is given by the following equation:
D = h * ln(2)/ln(c2/c1)
http://www.doubling-time.com/compute.php
7. Given your doubling time and the equation above, how many cells should you seed into a 6cm
plate in order to reach 50% confluency after 48 hours?