# Calculating cells/well in a 96-well plate - Calculating cells/well in a 96-well plate (Jan/29/2008 )

I'm having a serious brain fart. Could someone tell me how to calculate the following:

I need 5e4 cells/well in a 96 well plate, 50uL in the well.

Thank You!!!

-cellgirl-

Do you mean that you want to seed each well with that number of cells?
If so:
You want 5e4 cells per well in 50ul- so you want 5e4 cells/50ul which is 1e6 cells/ml (1000ul/50ul x 5e4)

-lauralee-

Yes, I want each well to have 5e4 cells. seeding in 50uL total.

I calculated by:
(5e4cells/well)(100 wells)(0.05mL/well)= 2.5e5cells/mL

That's 4 times less than what you got. What am I doing wrong?

-cellgirl-

QUOTE (cellgirl @ Jan 29 2008, 07:05 PM)
Yes, I want each well to have 5e4 cells. seeding in 50uL total.

I calculated by:
(5e4cells/well)(100 wells)(0.05mL/well)= 2.5e5cells/mL

That's 4 times less than what you got. What am I doing wrong?

Lauralee is right... you need 1x10^6 cells/ml

If you are calculating the required amount of cells per 96 wells plate
then

calculate enough for 100 wells
5x10^4 cells/wells x 100 wells
= 5x10^6 cells

calculate the volume of medium for 100 wells
50ul/wells x 100 wells
= 5ml

Therefore 5x10^6 cells in 5 ml of medium or 1 x 10^6 per ml.

Hope this may help.

-Minnie Mouse-

That's not quite right Minnie mouse- I think you made a typo and meant to write that you need 5x10^6 cells per ml.

Cellgirl- the first part of your calculation is right- ie to figure out the number of cells you need, multiply cells per well by the number of wells.
-so you end up with 5x10^6 cells in total for 100 wells
I am confused at your next step- I am not sure why you are multiplying by the volume you need per well?? You are effectively just dividing the number of cells by 20 (x .05 is the same as /20).
Ahhhh it is so hard to try and explain this over the net!! I can't seem to put into words how my brain figures this out!!

Minnie mouse explained it pretty well:

how many cells total do you need?? 5x10^6
what volume do you need?? 50ul x 100 wells= 5000ul (or 5ml)
so you need 5x10^6 cells in 5ml of media.

PS. Hopefully somewhere out there in the bioforum, someone has a logical way of explaining this so that cellgirl can get it right in her head for next time (and so I can steal your formula rather than doing the mental gymnastics my feeble brain usually has to do when working out calculations...)

-lauralee-

Thanks Lauralee... it is a typo

-Minnie Mouse-

Looks like I botched yesterday's experiment. I thought it looked wrong, but couldn't figure out why.
I multiplied the 100wells by 50uL to get the final volume including the cells/well (that atleast was my thinking process), but looks like instead I cut my cell density in 1/20

I'm with M.M. I wish there was a simple formula to use!

Any one out there know if there's one?

-cellgirl-

I don't think there's one single equation to use for this, but I can try to walk through this as plainly as possible.

You want to know how to get x many cells in y volume per well, and from that you want to know how many cells and how much volume for a 96-well plate.

In order to do this, one first must know the desired seeding density. This is done by taking the target number of cells, 5*10^4, and dividing it by the target volume in the well, in this case 0.05 mL. This gives us a seeding density of 10^6 cells/mL.

We can then use the equation (C1)(V1)=(C2)(V2), where C1=current concentration of cells in media, V1=current volume of media, C2=desired seeding density, to find V2, the volume of the seeding solution. Rearranging the equation to (C1)(V1)/(C2)=(V2) makes this a matter of plugging in the known values and doing the arithmetic (or plugging the values into a calculator and hitting enter).

So, we know how much of the seeding solution we'll end up with, based on how many cells we have. Finding how much of the seeding solution you'll need per plate is a simple matter of multiplying the volume in each well, 0.05 mLs, by the number of wells used, 96, which comes out to 4.8 mLs. This is the bare minimum amount of seeding solution that you will need to run this experiment on a full plate. Due to various sorts of systemic and equipment error, one never wants the exact amount needed for an experiment, so having at least 5mLs of seeding solution per plate would be advisable.

If one needs practice figuring this out, I'd suggest coming up with various plausible combinations of cell number and volume per well and running them through this setup until one understands where the numbers come from.

-TheSquire-

Hi cellgirl,

You can use my formula.

Number of cells required for 100 wells (96 wells plate)
= number of cells per wells x 100 wells
= A

The total volume of medium for 100 wells
= the volume of medium per wells x 100 wells
= B

Therefore you will need a total of "A number of cells" in "B volume of medium", then aliquot the required volume of cell suspension into each wells.

Don't worry... After a while, you will get used to the calculation and you can do it in your head.

You may wish to print the formula out and stick it at your bench.

Hope this may help.

-Minnie Mouse-