Homework SourseYour colleague in the laboratory was able to collect all his
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 and prepare a growth curve 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 Neubauer hemocytometer. You need to:
Show all work to receive credit.
a) Perform all necessary calculations to determine cell number (in this case cells/ml equals total cell number). Show all work.
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 Neubauer hemocytometer.
b) Make a Growth curve
| Day | Hemocytometer | Total Volume | PBS Dilution | Live | Dead | % Viability | Cells/ml (live cells only) |
| 1 | Neubauer | 1ml | NA | NA | NA | 95 | 1x10^5 |
| 2 | Neubauer | 1ml | 0 | 25 | 3 | ||
| 3 | Neubauer | 1ml | 0 | 64 | 6 | ||
| 4 | Neubauer | 1ml | 0 | 120 | 8 | ||
| 5 | Neubauer | 1ml | 1.5 | 230 | 6 | ||
| 7 | Neubauer | 1ml | 2 | 460 | 15 | ||
| 9 | Neubauer | 1ml | 3 | 673 | 10 | ||
| 11 | Neubauer | 1ml | 10 | 610 | 15 | ||
| 13 | Neubauer | 1ml | 6 | 401 | 45 | ||
| 15 | Neubauer | 1ml | 5 | 361 | 100 |
Solution
Day
Hemocytometer
Total Volume
PBS Dilution
Live
Dead
% Viability
Cells/ml (live cells only)
1
Neubauer
1ml
NA
NA
NA
95
1x10^5=10 x 104 cells
2
Neubauer
1ml
0
25
3
89.28
1.56 x 104
3
Neubauer
1ml
0
64
6
91.42
26.2 x 104
4
Neubauer
1ml
0
120
8
93.75
172.8 x 104
5
Neubauer
1ml
1.5
230
6
97.45
1825 x 104
7
Neubauer
1ml
2
460
15
96.84
19467x 104
9
Neubauer
1ml
3
673
10
98.53
91446 x 104
11
Neubauer
1ml
10
610
15
97.6
226981 x 104
13
Neubauer
1ml
6
401
45
89.9
38688 x 104
15
Neubauer
1ml
5
361
100
78.3
23522 x 104
To obtain cells viability= number of viable cells/ total number of cells
Cells number is in 1 mm. now convert it into 1 ml. 1 ml= 1mm3
So, for 2nd count; live cells are 25 in 1 mm;
In 1 mm3 = 253 = 15625 in 1 ml= 1.56 x 104 /ml
In dilution, multiply the number of cells by dilution factor after find cube of number of cells.
| Day | Hemocytometer | Total Volume | PBS Dilution | Live | Dead | % Viability | Cells/ml (live cells only) |
| 1 | Neubauer | 1ml | NA | NA | NA | 95 | 1x10^5=10 x 104 cells |
| 2 | Neubauer | 1ml | 0 | 25 | 3 | 89.28 | 1.56 x 104 |
| 3 | Neubauer | 1ml | 0 | 64 | 6 | 91.42 | 26.2 x 104 |
| 4 | Neubauer | 1ml | 0 | 120 | 8 | 93.75 | 172.8 x 104 |
| 5 | Neubauer | 1ml | 1.5 | 230 | 6 | 97.45 | 1825 x 104 |
| 7 | Neubauer | 1ml | 2 | 460 | 15 | 96.84 | 19467x 104 |
| 9 | Neubauer | 1ml | 3 | 673 | 10 | 98.53 | 91446 x 104 |
| 11 | Neubauer | 1ml | 10 | 610 | 15 | 97.6 | 226981 x 104 |
| 13 | Neubauer | 1ml | 6 | 401 | 45 | 89.9 | 38688 x 104 |
| 15 | Neubauer | 1ml | 5 | 361 | 100 | 78.3 | 23522 x 104 |
