2.1: Testing the Role of Extracellular Matrix (ECM) in Adherent Cell Culturing
(Determining Doubling Time)
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
Determine whether ECM choice impacts cell growth and/or survival
Vocabulary
Extracellular Matrix, adherent, hemocytometer
Outline
Trypsinize and count cells using the hemocytometer; distribute cells at specified densities to plates
containing various test substrates; assess cell attachment/growth/survival on the test substrates.
Materials
sterile
PBS, trypsin, 3T3 growth medium
two 6 cm uncoated plates
two 6 cm tissue culture treated plates
two 6 cm Matrigel 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
1. Prepare the microscope and assess confluency
2. Prepare the hood
3. 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 6-well culture plate.
4. Label the new culture plates.
a. In addition to the usual labeling, also label the 10cm plates with the quantity of cells to
be seeded (see later in the protocol).
b. Place the usual label information along one edge of the 6-well plate. Also label the lid of
the 6-well plate with seed values above each well (see later in the protocol).
5. Loosen the screw caps for the various (thawed) components
WASH AND TRYPSINIZE ADHERENT CELLS
6. Retrieve the cell culture plates from the incubator
7. Aspirate the old media from the plates
8. Add 5ml of prewarmed PBS to each aspirated cell culture plate
9. Aspirate the PBS from the plates
10. Add 1ml of prewarmed Trypsin solution to each 10cm plate. Scale the volume downward
accordingly for plates with smaller amounts of surface area.
11. While waiting for the trypsinization to occur, pipette 10 ml prewarmed 3T3 growth medium into
the desired number of fresh 10 cm plates.
12. Once the cell monolayer is mostly released from the plate surface, add 4mL of growth medium
to the plate.
13. 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. Slide layout
Figure 2. Neubauer Improved grid (detection area)
USING THE HEMOCYTOMETER
14. Obtain a fresh disposable hemocytometer.
15. Into an eppendorf tube, combine 20uL of cell suspension with 20uL of Trypan blue solution.
16. Load 10uL of the mix into one of the two sample injection areas (see Figure 1.)
a. Be careful to avoid introducing any bubbles.
b. Capillary action should help draw the solution into the detection area.
17. 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).
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.
18. 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.
b. When repeating the calculation remember to include the appropriate multiplier for your
additional dilution factor.
REPLATING TRYPSINIZED CELLS AT SPECIFIED CELL DENSITIES
19. Based on the concentration of your cell suspension 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:
3x105 cells
b. 10cm plate at:
105 cells
c. 6-well plate well at:
5x104 cells
d. 6-well plate well at:
2.5x104 cells
e. 6-well plate well at:
104 cells
f.
5x103 cells
6-well plate well at:
g. 6-well plate well at:
2.5x103 cells
h. 6-well plate well at:
103 cells
20. 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. If not all the cells are to be replated, dispose of the remainder by aspiration.
21. Gently rock the fresh plates to evenly distribute the cells.
22. Place the fresh plate(s) in the 37C incubator.
DAY 2 (Wednesday)
Protocol
1. Confirm attachment. Observe your cells under the microscope and estimate the % confluence
for each seeding condition.
2. From the 6-well plate, choose a well that is between 10% and 50% confluency, trypsinize the
cells from the well and use the hemocytometer to calculate the total number of live cells in
the well.
3. 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
1. Assuming that a 100% confluency 10cm plate contains 106 cells, determine the following:
a. How many cells are present per cm2?
b. How many cells should be used to seed:
i. A 10cm plate at a starting density of 20% confluency?
ii. A 6cm plate at a starting density of 20% confluency?
iii. One well of a 6-well culture plate at a starting density of 20% confluency?
2. Based on your calculated total number of live cells in one of the wells of your 6-well culture
plate, what is the approximate doubling time for NIH3T3 cells?
http://www.doubling-time.com/compute.php