Serial dilution of cells - (Apr/08/2013 )

I have 1.2 million cells/mL and I would like to bring it down to 0.5 cells/100uL for plating in a 96-well plate to create a stable cell line. What is the best serial dilution method to do this?

xbiol on Mon Apr 8 13:42:09 2013 said:

How many ml of your stock do you have? At this initial concentration of 1.5 million/ml, you can't 'serially dilute' it to get 0.5 (I guess it's also million eh)/100ul. What you can do is to concentrate your stock by centrifuging it again and resuspending in a smaller volume and then dilute to your final concentration and desired final volume using this formula: C1V1=C2V2

are you sure about your seeding density bec 0.5 million/well seems too much (so it's my mistake)...or if you really meant 0.5 cell/well...that's half a cell.......but then it would be like producing (selecting for) a positive hybridoma...

casandra on Mon Apr 8 19:56:07 2013 said:

Or maybe xbiol wants to do single cell seeding and has had too many wells with 2 cells in the past.... Then it's sometimes a good idea to use theoretical concentrations of something like 0,6-0,7 cells per well

To dilute your cells from 1.2x10^6cell/ml -> down to 0.5cell/100ul you'll need to do a several 1:100 (or 1:1000) dilutions first. Here is how I'll do it.

1.2x10^6cells/ml = 1.2x10^5cell/100ul (this way you have your stock and your desired final concentration in the same units).

Now, you could use C1V1=C2V2 but this results in having to take x10^-5 microliters which is pretty much impossible. So, here is where the several 1:100 or 1:1000 dilutions come in.

1. dilute your cells 1:1000 -> now you have a 1.2x10^2 cells/100ul
2. dilute this new stock 1:100 -> now you have a stock at 1.2cells/100ul

Now you can apply C1V1=C2V2 assuming you only need one plate and allowing for a bit extra I'll consider V2=10ml. C2=0.5cell/100ul and C1=1.2cell/100ul I'll let you do the rest

remember to prepare enough volume of your intermediate dilutions so you have enough V1 for this!!

Hoe this helps.