Titrating Antibodies on Human pbmc when staining cells with antibodies, background non-specific staining needs to be minimized. We titrate our antibodies to



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Titrating Antibodies on Human PBMC

When staining cells with antibodies, background non-specific staining needs to be minimized. We titrate our antibodies to determine the optimal antibody concentration yielding the greatest signal for the positive population and the lowest background signal for the negative population. We generally start our titrations at 2x the antibody manufacturer’s suggested volume or concentration necessary to stain 106 cells; next we perform four serial 1:1 dilutions from this starting point.

In this exercise, four antibodies stain ficolled human peripheral blood mononuclear cells (PBMC) cell surface antigens. These antibodies are Biolegend Brilliant Violet 605 Anti-Human CD4 (clone OKT4, catalog # 317438), Beckman Coulter PE-Cy5.5 Anti-Human CD3 (clone UCHT1, catalog # A66327), Beckman Coulter PE-Cy7 Anti-Human CD14 (clone RMO52, catalog # A22331), Beckman Coulter FITC Anti-Human CD16 (clone 3G8, catalog # IM0814U).

Procedure:

1. For all four antibodies, we will start the titrations at double the manufacturers’ recommended volume of antibody necessary to stain 106 cells.



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