To prepare for cell seeding, you washed GBM cells (grown in T-75 flask) with RT

Question

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.

Explanation / Answer

Each of the squares is 1 mm × 1 mm × 0.1 mm = 0.1 mm3 = 10-1 L = 10-4 mL

Total cells counted in 4 squares (i.e. in 4×10-4 mL) = 750

1. Average # of cells (i.e. cells per 10-4 mL) = 750/4 = 187.5

2. Dilution factor = 2

[because you diluted 50 L of cell suspension with 50 L of trypan blue, i.e. 1:1; dilution = 1/(1+1) = 0.5; dilution factor = 1/dilution - 1/0.5 = 2]

3. # of cells per L = 1875

(0.1 L contains 187.5 cells; therefore, 1 L will contain 187.5×10 = 1875 cells)

4. # of cells per mL = 1875000 = 1.875 × 106

(10-4 mL contains 187.5 cells; therefore, 1 mL will contain 187.5×104 = 1875000 cells)

5. Total number of cells obtained from flask = 5×1.875 × 106 cells = 9.375 × 106

(because total 5 mL suspension was obtained from the T-75 flask)

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