Question

1. (30 points) 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.

           

Day	Hemo- cytometer	Total Volume	PBS Dilution	Live	Dead	% Viability	Cells/ml (live cells only)
1	Fuchs	1ml	NA	NA	NA	95	1 x 105
2	Fuchs	1ml	0	25	3	89.3
3	Fuchs	1ml	0	77	9	89.5
4	Fuchs	1ml	0	132	4	97.1
5	Fuchs	1ml	1.5	222	9	96.1
7	Fuchs	1ml	2	415	16	96.3
9	Fuchs	1ml	3	643	19	97.1
11	Fuchs	1ml	10	659	14	97.9
13	Fuchs	1ml	6	410	24	94.5
15	Fuchs	1ml	5	389	86	81.9

Answer 1

2018 November October 2018 Saturday Total cells/ml - Total cous counted & dilation factor X10 No. of square cous/ml cell viability - Total viableceuiscunstained) /Total Cells(viablo + Dead)] x 100 Dilution Factor = Total Volume Cralume of sample et vallume - of dilution liquid) / volume of sample Total Valume=1mL (PBS used to wash the cell Dilution factor = 2 bellet, so that there is no dye No. of square = 5 present in the media) ceus/ml (live cous only) (225X2 X 104 = 1x15 Sunday 28 B. For Daya Totaal caus/ml = 25x2x109 = 1x109 3. For Day 3 Total coin me = 77XX.104 E 3.08x105 3. For Daya = Total ceus/mi z 132x3x109-5.28x105 1. For Day 5 ² Total cours/mi = 222X5X104 = 8.88 x 105 for Day 7 1 15X2 X10' = 1.66 X10° Total Cous/ml = 45

Sun Mon 29 Monday 2018 October Monday 6. For Day =) To-ldi ceurimi = 643X3 X 109 = 2.572X106 7. For Day 11 → Total ceus/mi = 659 x 3X104 = 2•636X106 8. For Day 13 = Total cous/mi = 410X3 X.104 =1:64 X106 9. For Day 15 = Total cousimi = 389xx104 - 1.556 X106 30 Tuesday Tuesday