Question

Your colleague in the laboratory was able to collect all his raw data from a growth curve on the Hela S3 cell line, but had to leave unexpectedly and has asked you to perform cell calculations to show your professor. This what your colleague did: he seeded cells into a 24-well plate (allows for replicate sampling and it is much easier to handle one 24-well plate rather than 24 flasks), and on each of the days indicated in the table on the following page, trypsinized and counted live/dead cells from three individual wells to allow for statistical accuracy. The information in the data table includes: day counted, media volume, dilutions, and number of live and dead cells counted (these numbers represent the average count from the three wells counted each day) with a Fuchs-Rosenthal hemocytometer. 

You need to 

Perform all necessary calculations to determine cell number (in this case cells/ml equals total cell number) 

Record of cell counts 

Please note that these counts have all been done with Trypan Blue Therefore, all cultures have been diluted two-fold (equal volume of cell suspension +equal volume of Trypan Blue). Take this into account when determining cell number. Any dilution listed in table is an additional dilution. Live and dead cells listed below represent the total number of cells in 5 large (1 mm) counting squares using a Fuchs-Rosenthal hemocytometer.

Answer 1

Answer:

Based on the given information:

• Hela S3 cells were seeded in 24-well plates
• Cells were trypsinized and live/dead cells were counted using Trypan blue dye.
• Numbers represent average count from three wells using a hemocytometer.
• Culture was diluted two fold (2x)
• 5 large counting square (1mm) of Fuchs-Rosenthal hemocytometer were used for cell counting (total number of cells).
• Area of single square = 1mm
• Volume of single square = 1 mm² x 0.1mm =  0.1 mm³ = 1 x 10^-4 ml
• Volume of 5 counting squares = 5 x 10^-4 ml
• Dilution factor for each sample = 2
• % Viability = 100*[Live cells / (live cells + dead cells)[
• Formula used: Cells/ml = [(Cell count in 5 squares)*2* PBS Dilution] / (5 x 10^-4)

Day	Hemocytometer	Total Volume	PBS dilution	Live	Dead	% Viability	Assay Dilution	Cells/ml (live cells only)	Cells/ml (live cells only)
1	Fuchs	1ml	NA	NA	NA	95	2	1 x 105	1 x 105
2	Fuchs	1ml	0	29	5	85	2	116000	1.16 x 105
3	Fuchs	1ml	0	74	8	90	2	296000	2.96 x 105
4	Fuchs	1ml	0	126	6	95	2	504000	5.04 x 105
5	Fuchs	1ml	1.5	232	9	96	2	1392000	1.392 x 106
7	Fuchs	1ml	2	430	12	97	2	3440000	3.44 x 106
9	Fuchs	1ml	3	623	14	98	2	7476000	7.47 x 106
11	Fuchs	1ml	10	650	17	97	2	26000000	2.6 x 107
13	Fuchs	1ml	6	421	35	92	2	10104000	1.01 x 107
15	Fuchs	1ml	5	364	99	79	2	7280000	7.28 x 106