Please answer q. 2 and 3 To prepare for cell seeding, you washed GBM cells (grow
ID: 178877 • Letter: P
Question
Please answer q. 2 and 3
To prepare for cell seeding, you washed GBM cells (grown in T-75 flask) with RT IX PBS and trypsinized the cells with 1 mL of 0.25% Trypsin-EDTA for 5 min @ 37 degree C. You then neutralized trypsin solution with 5mL of cell culture media. You transferred the 5mL cells in suspension to a 15mL conical tube, transferred 50mu L to a 1.5mL microcentrifuge tube, and mixed with 50 mu L trypan blue. You then transferred 10 mu L of the cell-trypan blue mixture to the hemocytometer and counted 750 cells from the four gray corner squares. Average # of cells = DF = Then it occured to you the cell stock is too concentrated which may affect count accuracy so you took 10 mu L from the initial cell:trypan blue mixture and mixed with 100 mu L of trypan blue, counted again and obtained a total of 250 cells from the four gray corner squares on the hemcytometer. What would be the of cells per Why is it necessary to wash cells with PBS before trypsinization?Explanation / Answer
Answer:
(2)
Original dilution with trypan blue = 2 times
Second time : 10 uL of diluted cells + 100 uL trypan blue = 110/10 = 11 times dilution
Total dilution factor = 2 X 11 = 22 times
Count from Hemocytometer (4 quadrants) = 250
Cells/mL = 240/4 x dilution factor x 10^ 4
= 60 x 22 x 10^ 4
= 13.2 x 10^ 6 cells/mL
= 13.2 x 10^ 3 cells/ uL
(3)
WAshing cells with PBS washes of the residual media which also contains serum. Serum has inhibitors for trypsin therefore washing with PBS removes the trypsin inhibitors.