calculation problem

[srve]

I am staining some cells with antibodies for FACS analysis.
my primary ab conc is 0.2ug/ul.

In one tube i have 1,000,000 cells (the approx volume of these cells is 40ul).i add 1ul of my primary ab to these cells and run my facs
If i have 10,000,000 cells (which is say 100ul of cells). How much of my antibody (ul) should i add to my 10,000,000 cells.

Is it 10ul that i add. Do i need to take into account the volume of my cells?

[kevin_a]

If the 1,000,000 cells in 40ul gives you good results, just dilute the 10,000,000 out to 400ul and use 10x the volume of antibody (10ul) at the same concentration - just as you asked.

You might not get the same level of staining or see higher background if you don't dilute the 10,000,000 cells out a little more.

Do you have to stain/run all 10,000,000 cells? If you can just use part of it, you'll save on the antibody.

[srve]

Hey Kevin,

Thanks for that. I have soem more questions. The volume of cells i start with is dependent on the number that are present, e.g 1,000,000 cell is approx 40ul and 10,000,000 cells in approx 100-120ul volume. if my stock is 2mg/ml and i add 1ul of this stock to my 1,000,000 (40ul) cells is the final concentraion of antibody in my cells 0.2ug?

If it is 0.2ug and i want to have the same concentration of antibody in 10,000,000 cells how much (ul) of my antibody stock do i add to my cells (which will be a volume of 100-120ul)

cheers

[kevin_a]

Well here is the real problem. You are trying to keep your antibody concentration the same, but your cells are much more concentrated, thus the amount antibody per cell is going to be lower. The conditions are not going to be same. That's why I suggested diluting your cells out from 100ul to 400ul and then adding 10ul of antibody - then all the concentrations will be the same.

If you really only care about the antibody concentration being the same do a ratio

1ul antibody / 40ul cells = x ul antibody / 100ul cells . . . solve for x

x ul antibody = 1ul antibody / 40ul cells * 100ul cells = 2.5 ul of antibody.

Thus adding 1 ul of antibody to 40ul or 2.5 ul of antibody to 100ul will give you the same final concentration of antibody when labeling the cells.
Concentration = 2mg/ml or 2,000ug/ul So, that gives you 2ug (1ul) per 40ul or 5ug (2.5ul) per 100ul = [0.05ug/ul] as the concentration in both samples.

Your cells on the other hand are very different [25,000/ul] (1,000,000 /40ul) vs [100,000/ul] (10,000,000 /100ul). The 4 fold difference might give you issues, so just be aware.