cell seeding calculation - (May/05/2011 )

Could you please help me in this calculation

I centifuged my cells and dissolved the pellet in 10 ml media.
In this 10 ml cell suspension I counted the cells and I have 0.5 mil cells/ml

I need 0.625 mil cells/ml in total volume 2,5 ml(in single well of 6 well plate)

Hope anyone here can help me with this calculation

Thank you very much in advance

-harry348-

hi..whats the tot vol 2? 5? or 2.5? i think i can help u

-uttess-

the total volume is 2.5ml

-harry348-

harry348 on Thu May 5 15:01:33 2011 said:

So, you have 0.5 mil/ml and need 0.625 mil/ml... You need to spin your cells down and resuspend in less volume.

Now, you can either spin them all, and resuspend so they are 0.625 mil/ml and then plate 2.5ml in your plate, or take an aliquot with the cells you need, spin, and resuspend in 2.5ml.

* For the first option you need C1V1=C2V2 where C and V are concentration and volume respectively.
In your case C1=0.5 V1=10 C2=0.625 V2=? V2= 8ml Spin down, resuspend in 8ml, plate 2.5ml.

* For the second option you need to think in absolute numbers. 0.625mil/ml x 2.5ml = 1.5625 million cells That's what you need. To take 1.5625million cells from your 0.5mil/ml = 1.5625/0.5 = 3.125 ml Again, as you only need 2.5ml, you'll need to spin down this 3.125ml and resuspend in final 2.5ml.

Personally, I'll spin down the lot, and resuspend them all at your desired concentration, irrespective of the volume you actually need for one well. Then just plate

-almost a doctor-

Think about it.....

if you only have 0,5 cells/ml (or 0,5milion cells, I dont use the "mil" here.. its the same logic)

then how can you ever obtain 0,625 cells per ml ......? you cant (by a simple calculation)
Or are you going to add cells without "liquid" ? ==> you need to remove liquid.

Are you sure you dont mean 0,625 cells in total ? Thus 0,625 cells in 2,5ml?

I am not sure is its just an theoreticall excercise or not.

If not you need to "remove volume" (as you cant "add cells without volume": do what almost a doctor says.

And the formula C*V=C*V, try to understand what it means or what it stands for.

-pito-

Thanks for helping frnds

-harry348-

to b clear to u about my question...
it is when i counted the cells in haemocytometer, the number of cells is 50 and the dilution factor is 1,
so now if we apply to the formula
No of cells per ml = 50*1*10000
= 0.5million cells/ml

So now what i need is like in a six well plate, in a total volume of 2.5ml i need 625000 cells/well.

What should I do now to get that count in each well?

Hope im clear now to you.

Thank you.

-harry348-

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...

-pito-

pito on Thu May 5 19:22:39 2011 said:

Thanx a lot Pito

-harry348-