How 2 Calculate cells/mL to cells needed per well? - EZ way to determine how much cell suspension to add/well for a certain number of (Aug/22/2006 )
There's has got to be an easier way to figure out what I need to do, than how I am doing it.
After I harvest cells, and count using a hemocytometer, I get about 120,000,000 cells/mL in my suspension. The suspension volume is 1mL.
I would like to plate my cells with a volume of 300ul, so that it will give me about 175,000 cells/well.
How can I calculate this?? I have a long way of doing it, but surely there's a better way.
i do like this
i count 1.000.000 cells/ml in my 1 ml suspension.
i want my final cell density to be 2.000.000 in a 5 cm dish.
so make it to the power of 10 like this
if 1x10 to the power of 6................in 1 ml
so 2x10 to the power of 6................x?
x=2 then x the volume of ur dish
so u will take 10 microns from ur 1 ml suspension and complete it by
4.990 of ur media in ur dish.
when I have to deal with concentration and volume I always use this formula :
where Ci is the initial concentration, Vi the initial volume, Cf is the final concentration and Vf is the final volume.
you want to know how many µL to take to have 175.10^3 cells per 300 µL ( = 175/0.3mL = 583.10^3 cells per mL)
then the volume to take is Vi = Cf*Vf/Ci = 583.10^3*300µL/120.10^6 = 1.46µL
other way to calculate :
you have 120.10^6 cells in 1 mL
so you have 175.10^3 cells in 1 mL/120.10^6*175.10^3 = 1.46.10^-3 mL = 1.46 µL
Just a small point :
Your cell suspension is far too dense. I was always taught to have between 50 and 100 cells/small square of the haemocytometer. This ensures that errors of counting do not occur.
Secondly you cannot accurately pipette 1.46uL of cell suspension. The cells should be diluted further and a larger volume added to each well.
Finally, everyone has their own way of calculating, find one that you are comfortable with and then stick to it.
I do agree with rhombus, that its better to have between 50 and 100 cells/small square. Gives less errors and more reliable.
I am not going to comment on how to count as I dont consider myself to b good at mathematics.
Just to be sure, I would check your calculations from the hemacytometer. They sound a little high for mammalian cells, assuming that is what your using, unless you concentrated your cells or were extreme perfusion.
The is how I would prepare a seed stock of cells at the concentration that you want for seeding, 175,000 cells/300 uL. I'll choose 20 mL for the seed stock volume for this calculation.
20 mL x (175,000 cells/0.3 mL) x (1/(120E6 cells/mL)) = 0.097 mL
Put 0.097 mL of your cell concentrate into 20 mL of your culture medium to prepare your seed stock, seed your wells using 300 uL.
divide the no. you want by the no. you have per ml e.g if you want 2 X 10^5 cells to seed a dish, and you have counted that you have 8X10^7 per ml . . . . .then 2X10^5/8X10^7 = the volume of cell suspension you need to use.