Counting and plating cells - (Feb/28/2012 )

Hi,

I need to count and re-plate cells.

In order to count cells with hemocytometer a serum free medium is needed, so after using trypsin and medium with FBS, I've used a centrifuge to take the medium out and tham put in PBS, and than counted the cells with hemocytometer and trypan blue.

Now, how do i plate the cells after that ?
At this point i have cells with PBS, but if i centrifuge the cells and take out the PBS and than re-suspend the cells in DMEM+FBS, i will change the cells concentration and the cell count will not be correct.

Is it essential to take out the PBS before plating the cells ?
Can i dillute the cells with DMEM+FBS without taking out the PBS ?

Firstly, I'm not sure why you have been told to count your cells in serum free media? I always count mine in my normal growth media, which contains NCS or FBS.

Secondly, centrifuging and resuspending your cells will change the concentration (assuming you resuspend in a different volume to the volume of PBS you used) but this won't make your cell count wrong. Once you have a concentration of cells, from your count, you just calculated the total number of cells- and this won't change no matter what the volume is.

E.g.
You have 5ml of a cell suspension, and according to your count, you have 2 million cells per ml.
2 million cells (per ml) x 5 (ml total)= 10 million cells total

Make sense?

Thirdly, it is not a good idea to simply add your media plus serum to the PBS as this will affect the concentration of the media and serum components. But if your PBS volume was small enough, and the media volume large enough, the effect may be minor enough to not matter. However for most applications I don't think the volume of media you are adding would be enough to negate the diluting effect of the PBS.

Fourthly, if you really do need to count your cells in serum free media, I would suggest using DMEM (serum free) rather than PBS to resuspend your cells. That way, when you are topping up your volume with DMEM+FBS to plate your cells, only the serum concentration will be out. You can then simply calculate how much extra serum you should add and adjust.

Hope that helps

Thanks

I will take the cells volume that i need in PBS according to the cell count and than centrifuge, then take out the PBS and add DMEM+FBS.
The concentration maybe will change, but the cell count will stay the same.

I have another question.
Does leaving the cells for a long time in PBS (1-2 hours) will harm them ?
because that i have a lot of cells to count and it may take a while before I count all the plates.

In theory, no it shouldn't hurt them, but in practice, they will become depleted for a range of compounds that are metabolized (e.g. sugars), which may or may not affect how your results turn out. If you keep them on ice while counting, they should last OK.