Flow cytometry-antibody conc. - (Jun/21/2012 )

Hi there,

I'll be doing FC for the first time next week, and I have a protocol but I'm slightly confused about one thing...after I resuspend my cells in PBS/BSA it tells me to take 200uL of cells and incubate with 1:200 of primary antibody...so does this mean if I have 200uL of cells, I add 1uL of my antibody...and then if I wanted to to another conc of antibody say 1:400 I would add 0.5uL of antibody...I recall being told I would have to do serial dilutions of my primary antibody..but how do I go about doing serial dilutions..if i need 200uL of cells to start with?

Hope I make sense!
cheers,
b_06er

Can't you do your serial dilutions before you resuspend the cells in the PBS/BSA?

Your idea is basically correct. If you have the cells in 200 microliter and the antibody concentration you want is 1:200, you add 1 microliter antibody and so on.

Has someone from your lab done the antibody titration? Maybe when you were told to do serial dilution of the antibody, what they meant was to do antibody titration. This means you test out the optimum amount of antibody you need to use per sample. So you start say 1:50 and go down with the amount till say 1:800 and see which concentration gives you the optimum signal (not too low and not too high). Once you figure that out, you just use the optimum concentration for all your experiments from now on