I would try to simplify the procedure. All you really need to know is how many cells are needed to seed each well (this may be an educated guess, unless you want to try a few different values), the number of cells per unit volume in your homogentate and the volume of homogenate that will give the required cell number. You may have to dilute your homogenate or resuspend it in a smaller volume after centrifugation.

Hello Maya Hoon

You are right. You can also do the calculation as follows:

50,000 cells per well per ml is required.

You have 2,60,000 cells ( 13 x 10000 x 2 )

So by the formula

N1V1=N2V2

Where

N1 = Total number of cells = 2,60,000

V1= the volume to be calculated X

N2= Number of cells per well = 50,000

V2= Volume per well of the 24-well plate = 1ml

Therefore ,

2,60,000 x X = 50,000 x 1

X= 0.192 ml for one well per ml.

So for 216 wells the required number of cells will be 216 x 0.192 = 41.5 ml.

While taking the cell count using the hemocytometer you should use lesser volume of cell suspension (say 10ul and not 100 ul). So 10ul of cell suspension and 10ul of trypan blue.

You should always calculate 2 wells extra because of the volume effect. The last well will not receive the required volume and there is a possibility of an error.

Best Wishes.

Malcolm Nobre Thank you very much for the thorough explanation! This simplified equation is definitely extremely helpful.

This may not be a good question, so I apologize in advance if this is obvious or a poor line of thought, but we do not have a clear protocol written out in our lab, so I would like to write one out so that it is more clear for our labmates and easily replicable. My follow-up question is as follows:

My cell suspension I would take the 10 ul from would have ~10 mL in it. So to reach the 41.5 mL of cells, would I have to passage more flasks (say 3 more flasks with the ~10mL of suspended cells as a product each) to get that 41.5 mL of cells? Or do I do something entirely different?

Thank you so very much.

Hi Maya Hoon

You will have to do something that is entirely different. The total number of cells required for nine 24-well plate is more than what you have. You need 10,800,000 (216 x 50000) cells for 216 wells if you add 50,000 cells per well. Your count comes to 2,60,000 cells/ml.

So, you will have to prepare more flasks so as to obtain more cells than what you actually need. If you have a rough idea of the number of cells you can obtain from say one T-75 flask, then according plan to seed cells in that many flasks so as to get the required number of cells.

Pool the cell suspension from all the flasks in two or more 50-ml centrifuge tubes and centrifuge. Add 1ml complete medium to all the 50 ml tubes, tap the cell pellet and pool the cells in one 50-ml centrifuge tube. Add 1ml complete medium to that 50-ml centrifuge tube and take the cell count. Use 10ul cell suspension + 10ul trypan blue, mix and take the count with hemocytometer. Then calculate the cell count as you have done above with dilution factor 2. If the cells are too crowded to count then increase the dilution factor. Whatever count you obtain say X, it will be X/ml.

Then use the formula I mentioned above that is N1V1=N2V2.

I hope this will be helpful.

Good Luck.