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:
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
Actually in a Haemocytomer/ Neubauer chamber, four functional chambers are there. Blood cells are counted in these 4 chambers and the aerage is considered. The average multiplied with 10^5 is the cell number/ ml according to hemocytometer formula. If 10 times dilution is done with PBS this no. is multiplied with 10 for per ml cell no.i.e the dilution factor (if diluted) is always multiplied to get cell number/ml. So, the number of cells will be-
A. Day 2- 25x10^5 cells/ml
Day 3- 64x10^5 cells/ml
Day 4- 120x10^5 cells/ml
Day 5 -230* 1.5= 345x10^5 cells/ml
Day 7- 920x10^5 cells/ml
Day 8-2010x10^5 cells/ml
Day 9- 2019x10^5 cells/ml
Day 11-610x10^6 cells/ml
Day 13- 2406x10^5 cells/ml
Day 15-1805x10^5 cells/ml
b) For making the growth curve, plot no. of days in X-axis and no. of cells (above mentioned cell numbers) in Y-axis. Since there is no option for figure the growth curve could not be shown on this page.
