Antibody titrations and total cells to be stained

[TroyLim]

Hi I am a new user, and am planning to use CyTOF to analyse circulating immune cells of leukaemia patients recruited into clinical trials. As i am hoping to identify rare populations i hope to obtain as much events as i can (hopefully 500000 events per sample).

When titrating my antibodies by staining 3 x 10^6 cells most of my antibodies work well at 1:100 titration (my 30+ antibodies are 80% preconjugated, 20% conjugated in house). However in order to save on antibodies, i tried using the same titrations when staining 10x10^6 cells, and found similar protein expressions compared to when staining only 3x10^6 cells.

I know fluidigm recommends to uptitrate every 3x10^6 cells stained, but i wonder if anyone has any experience in using same antibody titrations for a larger cell volume? Or is it the consensus that CyTOF antibodies are very sensitive to total cell required to be stained?

Thanks for your help.

[taxkourel]

That sounds about right. Depends on the target protein too. You can probably save on antibodies if you titrate your antibodies upfront in your sample of interest before starting ur experiment. I ve used some at 0.125x and they stained as well as at 1X. This will also help you identify spillover on adjacent channels, oxides etc so always a good idea. Finally, half a million events may be enough but focus on how many usable events you get in the end (ie live singlets, if looking at immune cells cd45+ etc) since your usable events might end up being much less and you ll need to shoot for a higher number of acquired events. Good luck!