JC-1/Jerry/Tomoko/UCSF

In Vitro Study: U87MG FL Growth Curve

Aim: To determine the doubling time of the U87 cell line.

Cell Line: Human GBM Cell Line U87 Experimental design:

Cell Seeding1 x 10E5 cells/5mL

(Day 0, 8/18/14) (Day 2, TP1) (Day 3, TP2) (Day 7, TP3) (Day 11, TP4)

Material: U87FL Cell Line T25 Flasks (10) Media (DMEM-H21, 10% FBS, 1% Fungizone/Penstrep/NEAA) Trypsin (0.05%) Trypan Blue (0.4%) 50 mL Falcon Tube 5 mL Round-bottom Tube

Procedure:1. Seed 10 T25 flask with 1 x 10E5 cells on Day 0 (8/18/14).2. Starting with time point 1, repeat the following steps at each time point with two flasks.3. Aspirate the media from the flasks.4. Wash the flasks with 1 mL trypsin and aspirate it.5. Let the flasks sit in 1 mL trypsin and detach all cells.6. Neutralize the flasks with 4 mL media (total volume of each flask is now 5 mL). Important: Make sure to

produce a single cell suspension.7. Put 100 µL sample and 300 µL trypan blue into 5 mL round-bottom tubes for each sample. (Dilution Factor=4)8. Place 10 µL of mixture from one test tube into the hemacytometer.9. Count the number of cells on each side and average them (Cell Counted Average x 10E4 x 5 mL).10. Clean the hemacytometer.11. Repeat steps 8, 9, and 10 for the second test tube.12. Average the number of cells counted for the two flasks for the final cell count for the time point.13. Clean up and dispose of all materials properly.14. Plot the resulting time point data on a growth curve.

Work: Seeding: Average Cell Count x 104 x Original Sample Volume = 14 cells x 104 x 10 mL = 1.4 x 106 cells

- Two flasks with 50% confluent were counted before adjusting the appropriate volume for seeding. Timepoints: Average Cell Count x 104 x Original Sample Volume = TP1: 8 cells x 104 x 5 mL = 4 x 105 cells Generation Time = G = t/n = t/[(logB1-logB0)/log2] = 24/[(log(6.5x105)-log(4x105))/log2] = 44.77 hours

- t = Time; B0 = Initial Cell Count; B1 = End Cell Count; n = Generation Number

Results:

Day Time Point Cell Count Total Volume Total Cells/T25 Average Total Cells

2 – 8/20/14 1 Flask 1: 8 x 104/mL 5mL 4 x 105

4 x 105

Flask 2: 8 x 104/mL 5mL 4 x 105

3 – 8/21/14 2 Flask 1: 10 x 104/mL 5mL 5 x 105

5.8 x 105

Flask 2: 13 x 104/mL 5mL 6.5 x 105

7 – 8/25/14 3 Flask 1: 126 x 104/mL 5mL 6.3 x 106

6.2 x 106

Flask 2: 121 x 104/mL 5mL 6.05 x 106

11 – 8/29/14 4 Flask 1: 119 x 104/mL 5mL 5.95 x 106

6.2 x 106

Flask 2: 127 x 104/mL 5mL 6.35 x 106

Conclusion:Time points one through four have provided data that maps out a growth curve reaching the stationary

phase (where growth plateaus). My data shows a 28.08 hours doubling time. This generation time was calculated by using coordinates obtained from Prism. After plotting the graph, I used the coordinates of 1x106 cells at 3.71 days and of 2x106 cells at 4.88 days. The cells were in log phase from Day 0 to Day 7, and they reached the plateau phase by Day 7 (the cells became confluent). If performed again, this experiment could be improved with more time points for a more accurate representation of the actual growth curve.