# Basic help in cell counting...... - (Sep/22/2007 )

Hi all,
I have a small doubt thats been bothering me for long. I am doing proliferation assay using ten 96 well plates and i seed 2000 cells per well with 50ul of media per well. In order to that I need 50ml of media in total (48 actually, just making it more just in case!!). So to seed the cells per well, I count the cells using haemocytometer. I usually count the four corner squares and the middle square and use the fomula average of 5 squares/0.02 * 1000, to get the cell density per ml. Please note that I dont use trypan blue or what ever, i just resuspend the cell pellet after centrifugation in 1ml of media and take 10ul of that and count the cells. If i get 3 million cells per ml after the calculation, does that mean the total number of cells that i have is 3 million or do i need to do more calculations? Also since i need 50 ml media and every 50ul of it should have 2000 cells, do i need to have 2million cells in 50ml? and to calculate the 2 million cells do i have to divide 2million by 3 million to get amount (in ul) to take from the 1ml cell suspension and add to the 50 ml media? so that every 50ul of the 50ml cell suspension has 2000 cells??? Please help.. I hope i have made myself clear.. Thanks...

-molecular_medicine-

If you use an average of 5 sq then the factor should be 10,000. Your cell concentration should be 10,000 X the number you get. Say if you have an average of 300 per sq, your cell concentration is 3,000,000/ml.

If you have 3 mil/ml and you want 2 mil, yes, you need 2/3 ml = 666ul

2000/50ul*50,000 ul= the cell number you need for 50 ml of working solution: you need 2,000,000 cells total.

The other way to estimate will be you have 100 wells/plate, each well you need 2000, then you need 200,000 total cells/plate, and you have 10 plate to seed, that will be 2,000,000 cell in all.

-genehunter-1-

Technically you should subtract the volume that you took out of your 1.0 ml to calculate the total cell number, but in this case you don't actually seem to need the total cell number so it isn't important.

-bob1-

Since I'm not very good in calculations I always use a "proportion" (not sure if this is the proper english name):

Ci : Vi = Cf : V

Where:
Ci: cell density in your initial suspension (ie: 3.000.000/ml)
Vi: volume of your initial suspension (ie: 1ml)
Cf: number of cells you need to have your final suspension (ie: 2.000.000)
V: volume of initial suspension (O,666 ml) that contains the number of cells you need (2.000.000) and that you have to take and resuspend in medium

So it will be V= (Vi x Cf)/Ci
Since you use Vi=1ml it's Cf/Ci which is, as you said, 2.000.000/3.000.000

If you have to resuspend your pellet in more than 1ml, this "formula" is quite useful. Be careful that if you use Vi in ml also V will be in ml. So if you prefer working with ul, Vi=1000.

I hope I explained myself!

-panda-

QUOTE (molecular_medicine @ Sep 22 2007, 11:04 PM)
Hi all,
I have a small doubt thats been bothering me for long. I am doing proliferation assay using ten 96 well plates and i seed 2000 cells per well with 50ul of media per well. In order to that I need 50ml of media in total (48 actually, just making it more just in case!!). So to seed the cells per well, I count the cells using haemocytometer. I usually count the four corner squares and the middle square and use the fomula average of 5 squares/0.02 * 1000, to get the cell density per ml. Please note that I dont use trypan blue or what ever, i just resuspend the cell pellet after centrifugation in 1ml of media and take 10ul of that and count the cells. If i get 3 million cells per ml after the calculation, does that mean the total number of cells that i have is 3 million or do i need to do more calculations? Also since i need 50 ml media and every 50ul of it should have 2000 cells, do i need to have 2million cells in 50ml? and to calculate the 2 million cells do i have to divide 2million by 3 million to get amount (in ul) to take from the 1ml cell suspension and add to the 50 ml media? so that every 50ul of the 50ml cell suspension has 2000 cells??? Please help.. I hope i have made myself clear.. Thanks...

I'm confused, why do you divide 5/0.02 ?
I count 5 squares and then multiply the number i get x5x10,000 that's your cells/ml. so if your total volume is 1ml, that's your total cells.
if you want to have 2000cell/50ul, yes you do need 2,000,000cells in 50ml. so divide 2 by your cell/ml, take that volume and bring it up to a final volume of 50 ml.

-almost a doctor-

Almost fainted, I´ve been using the average of 4 corner squares *10.000 for as long as I can remember. The division by .02 had me mystified, it actually overestimates your cell number by a facor of 5.

I liked PANDA´s method, I guess I go by the same way, but I take a less automatic approach. Since I usually get a un-even volume, I measure it as closely as possible (say 855 ul measured by pipette man, you can twist the volume node until you get the exact volume, then you check by pipetting up and down a couple of times and not getting any bubbles).

Then: say average of 8 corners (both side of the hemocytometer)=25 *10000, (250.000 cells per ml)*0.855=213.000 cells in total volume. and from here you can adjust your volume and the number of cells you want. Say you want to seed 10.000 cells in 100 ul? You divide 213.000cell/855ul gives you 249 cells/ul, 10.000 cells/40 ul. From here you can either bring your volume up in order to make your dilution 100cell/ul (you have to bring it to a final volume of 2137 ul by adding 1282 ul of media), or you can pipette 60 ul into each well and add the 40 ul that include your 10.000 cells.
Best of luck

-Alejandro-

I agree with Alejandro and Panda, that is the method/approach I also use. I too was astounded the incorrect methods described here!!!

But don't believe us, go out and find yourself a quality cell culture book and look it up for yourselves!!!

-lauralee-