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Galina Rupchina Galina Rupchina
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5 months ago
You wish to set up a multi-well cell culture experiment and begin by trypsining a T75 flask of adherent cells with 2ml of trypsin. After adding 3ml of media to this and transferring the suspension to a sterile tube, you carry out a cell count. To check cell viability at the same time, you mix 50μl of the cell mix and 50μl trypan blue and incubate this for 5 minutes. You then load two chambers of a cell countess side and count the number of blue and clear cells. You wish to set a multi-well cell culture experiment and begin by trypsining.

The count is as follows:

Reading 1: 49 clear cells and 3 blue cells

Reading 2: 43 clear cells and 2 blue cells

1. Calculate the number of viable cells and express this in cells per ml e.g. 2.76 x 105 cells per ml.

2. What percentage of cells are non-viable?

3. Calculate the total number of live cells in the tube.
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wrote...
Educator
5 months ago
I'm not sure if this is correct.

Please let me know your thoughts
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Galina R. Author
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5 months ago
Thank you so much for the help!

Your approach is in the direction I was heading myself so I think it is alright.

The question has two more points and I would appreciate it if you could share your thoughts about how to solve them.

4. You now need to seed three wells of chamber slides such that each well contains 5,000 cells in a volume of 1 ml of growth medium in each well. 

5. Calculate the volume of cells and the volume of fresh medium that you should dispense into a sterile tube to make up 4 ml of a 5,000 cells/ml viable cell solution.
wrote...
Educator
5 months ago
Galina, I'm reluctant to answer further because I cannot verify if what I did above is correct. If 3 mL of medium is added to the cell suspension, then maybe the dilution factor is:

DF = 3 mL / 50 μL = 3 mL / 0.05 mL = 60.

But I'm not too sure if 2 mL of trypsin also adds to the 3 mL, making it 5 mL in total. If that's the case, then we have:

DF = 5 mL / 0.05 mL = 1000

The dilution factor is important because when we multiply by 10^4, we must also multiply by the dilution factor.

Thus, for #1, it could be:

Viable cells = 46 the average * 60 the dilution factor * 10^4 = 2.76 * 10^7 cells / mL, but again I'm not certain. If that were the case, then we can say that there are 2.76 * 10^7 cells / mL in 3 mL of medium.

Would you happen to have a sample question I could use to confirm my thinking?
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