Is it 625.000 cells per ml or per well???? Because you are giving other information now...

If it is 625.000 cells per well and you have 6wells, then you need to have 3.750.000 cells in total.

And those cells will be in 15 ml (because each well is 2,5ml and 6 wells, makes 15 ml).

(I am asuming its 2,5ml per well and not 2,5 ml in total for 6 wells.. this would be odd anyway, because you cant divide 2,5by 6 ..., it doesnt give a number you can use as a volume in your pipette)

So you need to have a concentration of: 250.000 cells/ml (3.750.000 cells/15ml).

Your stock is 500.000 cells/ml , but you need 250.000cells/ml ... so what do you do?

Either you just add another 10ml so you have 250.000cells per ml (1/2 dilution).

And enough to fill the entire well (you have 2Oml and only need 15).

Or if you dont want to use the entire stock dilution and just want to have 15ml, you use the C*V=V*C formula like this:

500.000 cells/ml (stock concentration) * V? (volume you need to take from stock to get the desired volume/concentration) = 15ml (wanted end volume) * 250.000cells/ml (wanted concentration) and V is= 7,5ml and this you take from the stock and you then fill it untill you reach 15ml (add clean medium)

Try to find the logic behind the formula.. dont just remember it by heart.

The second option , I dont like it, because you have just enough to fill all the wells.. you cant afford to spill a bit.. and we all know that if you take 1O ml to divide over 10 wells (1ml each) that the last well will be a bit less then 1ml.. so always take (prepare) a bit more then the working (end) volume you need.

So you can take for example 17ml or 16 wanted volume (try the formula yourself to see if you get it..)

Or just take option 1 and add 10 ml to your stock of 10ml

And about the dilution factor with the haemocytometer, are you sure its 1 ? And not that you did a 1/10 dilution? Because a dilution factor of 1 means nothing: it means you didnt dilute at all... I would find it weird to use that...

**Edited by pito, 05 May 2011 - 11:24 AM.**