Cell Proliferation Assay

 

Objectives

·        Quantitatively assess the effects of serum on the growth and replication of HDF cells.

·        Understand and calculate cell doubling time.

 

Materials

24-well TC treated plates

DMEM with 1% serum and 1% antibiotic

DMEM with 5% serum and 1% antibiotic        

DMEM with 10% serum and 1% antibiotic      

Materials as required by cell passaging procedure

 

Safety

5.      Wear disposable latex gloves at all times.

6.      Wear safety glasses at all times.

 

Test Conditions

A.     DMEM with 1% serum      

B.     DMEM with 5% serum      

C.     DMEM with 10% serum    

 

	Total #		# Wells in Specified Conc.
	Wells	1% serum		5% serum	10% serum
Plate on Day 1	33	33
Count on Day 1 - 4 hrs	6	6
Count on Day 3	9	3		3	3
Count on Day 6	9	3		3	3
Count on Day 8	9	3		3	3

 

Ø      Initial seeding density tested on Day 1 - 4 hrs.

Ø      Test samples incubating in 1%, 5%, and 10% serum tested on Days 3, 6 and 8.

 

Prepare Cells (accomplished by T.A. or Instructor)

1.      Proceed through cell passaging protocol until step #15. 

2.      After centrifugation, resuspend the cells in 15 mL DMEM with 1% serum. 

 

Experimental Summary

 

	Plate cells	Cell Count	Change/Replenish Media
Day 1	X	X (seeding estimate & 4 hr sample)	X (Days 3, 6 & 8 samples)
Day 3		X (Day 3 sample)	X (Days 6 & 8 samples)
Day 6		X (Day 6 sample)	X (Day 8 sample)
Day 8		X (Day 8 sample)

 

Plate cells (Day 1)

1.      Determine the cell concentration using the Coulter Counter.  Prepare three vials to test, each with 500 mL cells and 9.5 mL of Isoton.  Remember to mix cell suspension before aliquotting cells to Coulter Counter vials.

2.      Lay out and label 24-well TC-treated plates with different conditions. 

3.      Determine the total volume of cell suspension that is to be seeded. Each well should be seeded with a volume of 1 mL of cell suspension. You will need an additional 3 mL for step 10. Always prepare a little extra. 

4.      Complete the calculation for dilution of the cell suspension to be used for plating at 5,000 cells/mL. Confirm your cell dilution calculations by checking with a TA.

5.      Dilute cell suspension (from stock) in DMEM with 1% serum to 5,000 cells/mL.  A 50 mL centrifuge tube should be adequate to hold the necessary volume of cells.

6.      Gently mix cells (pipet mixing or gentle inversion of tube).

7.      Seed 1 mL of cell suspension into each test well on your plate.

NOTE:  When seeding, mix the cells gently and often (every 6 wells or every 15 sec) to prevent settling of the cells. Cells settle VERY fast;  if different numbers of cells are aliquotted to the wells, your results will be difficult to interpret.

8.      Place plate in incubator for at least 4 hrs. 

9.      While the plate is in the incubator, prepare 3 Coulter Counter vials, each with 9 mL Isoton.  Aliquot 1 mL of the remaining diluted cell suspension into each vial.  Determine the cell concentration using the Coulter Counter.

 

Initial Exposure to Different Growth Conditions (Day 1)

1.      Remove the cells from the incubator after 4 hrs.

2.      Check cells under microscope to estimate the fraction attached. 

3.      Using a glass Pasteur pipet, aspirate the liquid media covering cells in wells. 

4.      Add 1 mL of DMEM containing 1%, 5%, or 10% serum, as needed, for sample wells for Day 3, Day 6, and Day 8. 

NOTE:  STEPS 3. & 4. SHOULD BE DONE SEQUENTIALLY FOR EACH TEST CONDITION.

5.      Determine the number of cells attached after 4 hrs incubation using the directions for “Cell Number Determination” below. 

6.      Return plate to incubator.

 

Cell Number Determination (Days 1, 3, 6 and 8)

NOTE:  This procedure is conducted at Day 1 - 4 hrs, Day 3, Day 6, and Day 8.

NOTE:  Six samples are prepared for Day 1 - 4 hrs since students often have a difficult time collecting reliable data at this time point.  A minimum of three reliable counts is necessary.

1.      Remove cells from incubator.

2.      Check cells under microscope.   Estimate the cell density.

3.      Using a sterile glass Pasteur pipet, aspirate the liquid media covering the cells in the wells you wish to determine the cell number. 

4.      Add 1 mL of PBS.  Swish around.  Aspirate liquid.

NOTE:  In this and subsequent steps, when adding a liquid to a well, squirt the liquid on side wall of well rather than in center of well.

5.   Add 250 mL of trypsin to each well. Lightly swish trypsin.

6.      Place plate in incubator for 5 min.

7.      While waiting for cells in incubator, prepare Coulter Counter vials with 9 mL Isoton. 

8.      Remove plates from incubator.  Tap on side of plate several times. Be careful to not spill the wells with 1 mL liquid.  Check cells under microscope to confirm that cells are detached from the surface.  Failure of cells to detach is a common mishap in this protocol.

9.      Add 750 mL DMEM with 10% serum to each well.

10.  Do the following for each well, one at a time:

a)      With micropipette set at 900 mL, gently suck the liquid up and down three times. 

b)      Aliquot 900 mL of cell mixture to a Coulter Counter vial with Isoton. 

c)      Add 900 ml Isoton/cell mixture back into the well. 

d)      Repeat steps a) and b).

e)      Repeat steps c), a), and b).

f)        Move remainder of liquid in well to Coulter Counter vial.  Failure to retrieve all the cells from the vial is a common mistake in this protocol.

11.  Repeat for each well of the Cell Number Count test.

12.  After removing cells, do a visual check with the microscope to make sure that few cells remain on the well surface.

13.  Return plates to incubator.

14.  Determine the cell number/concentration using the Coulter Counter.

15.  Dispose of biohazardous wastes appropriately.

 

Media Replenishment (Days 3 and 6)

1.      Remove cells from incubator.

2.      Check cells under microscope. 

3.      Using a sterile glass Pasteur pipet, aspirate the media covering the cells. 

4.      Add 1 ml of DMEM containing 1%, 5%, or 10% serum, as needed.

5.      Return plates to incubator.

 

Calculations/Questions

NOTE:  In generating plots and doing calculations, remember that “Day 1” is the start of the experiment (time = 0), “Day 3” is 48 hrs after Day 1, etc.

1.      (20 pts)  Plot the cell number as a function of time for the different culture conditions (i.e. serum concentrations).  Use a logarithmic axis as appropriate. 

2.      (20 pts)  Within the test condition of 10% serum, are the cell numbers at Days 3, 6, and 8 statistically different from one another?  What type of statistical test did you use?

3.      (20 pts)  At Day 8, are the numbers of cells exposed to 1%, 5%, and 10% serum statistically different from one another?  What type of statistical test did you use?

4.      (20 pts)  Are the cells in exponential growth?  Growth rate is often expressed as a doubling time.  Determine the cell doubling time for each of the culture conditions.  Explain what data was included to make each doubling time calculation and why.  Comment on the doubling times in light of the different serum concentrations.

5.      (20 pts)  Are your results consistent with the data collected from the Anti-PCNA Staining assay?  Discuss the expected relationship between the two assays, and how your data is or is not consistent with that relationship.

6.      (10 pts)  Compare the initial attached cell density measured 4 hrs after seeding (Initial Exposure, Step 5) with a “theoretical” cell density (Plate Cells, Step 10).  If they are dissimilar, hypothesize an explanation.

 

Answers to Calculations/Questions should be recorded in your laboratory notebook.  The point values are listed above.