I have new antibodies:CD40/PDL1/CD206/CD68/CD163 and need to perform the antibody titration. These markers are going to be use for Human PBMC in the experiment. I would like to ask: Do I need to activate them for the purpose of getting the positive signal? Our lab has U937 cell line, will it be fine using this one to titrate the antibody or will it influence the result on the human PBMC sample I will be running after the optimization? Pleae advice. Thank you!
Hello , To titrate antibodies you do need positive binding to be able to evaluate the staining index - https://www.biolegend.com/en-us/blog/the-stain-index-what-is-it-and-what-does-it-tell-you. I don't see why you could not use species appropriate compensation beads as surrogates? The CVs on the negative beads is likely to be smaller than on cells but they have a defined bead binding density against which you should be able to titrate your antibodies and determine an optimal antibody concentration for experimental samples.
Morning, Thermo fisher has various bead sets https://www.thermofisher.com/uk/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometry-calibration/flow-cytometry-compensation-tools.html#selection-guide
Compensation beads are coated with a density of antibodies with various reactivity to commercial reagents. You should have negative and positive staining beads from which to evaluate a staining index. If you can't easily generate positive cells to titrate you new antibodies against. Best wishes
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