Hamocytometer grid # of cells/nal-(Average) (DF) (10.) If multiple DFs, # of cel

Question

Hamocytometer grid # of cells/nal-(Average) (DF) (10.) If multiple DFs, # of cellsmL-average) (DFI ) (DE ( DFn) ( i 0") # of cells). L-(Average) (DF) (101) If multiple DFs # ofcellshl. = (Average) (DFI) (DF2) (DFn) (10') (1) To prepare for cell seeding, you washed GBM cells (grown in T-75 flask) with RT IX PBS and trypsinized the cells with l mL of 0.25% Trypsin-EDTA for 5 min @ 370. You then neutralized trypsin solution with 5mL of cell culture media. You transferred the 5mL cells in suspension to a l 5mL conical tube, transferred 5OuL to a 1.5mL microcentrifuge tube, and mixed with 50? L trypan blue. You then transferred 10? L of the cell trypan blue mixture to the hemocytometer and counted 750 cells from the four gray corner squares. Average # ofcells- . DF . # ofcells per ?L- . # ofcells per mil- . The total # of cells obtained from the T-75 flask (2) Then it occurred to you the cell stock is too concentrated which may affect count accuracy so you took 10? L from the initial cell:trypan blue mixture and mixed with 100? L of trypan blue, counted again and obtained a total of 250 cells from the four gray corner squares on the hemocytometer. What would be the # of cells per ?L? (3) Why is it necessary to wash cells with PBS before trypsinization?

Explanation / Answer

Ans:

1.

(2) Cell will be 75 cells /ul because the dilution factor of trypan blue is 1:25 with cells.

(3)Cells are washed first with PBS because most of the growth media for cell culture contains Fetal Bovine Serum (FBS). FBS contains protease inhibitors, such as ?1-antitrypsin and ?2-macroglobulin. These inhibitors stop the trypsin protease to act on. So we need to wash cells with PBS t get rid of any serum of growth medium where cells were suspended for privious passaging. So we have to wash with PBS after trypsin also for the same.

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