How can the ideal concentration of an antibody for primary labeling be determined?
Different concentrations of a fluorochrome-conjugated primary antibody are used for primary labeling. The following recommendations can be used as a guideline: • Perform a log2 dilution series starting with 2 µg/100 µL, 1 µg/100 µL, 0.5 µg/100 µL down to 0.016 µg/100 µL. • Suspend 106 cells in 50 µL of buffer and add 50 µL of the antibody dilutions. • Incubate for 10 min at 4–6 °C. If an unconjugated or biotinylated antibody is chosen for primary labeling, an additional staining step has to be applied before flow cytometric analysis. The second labeling step is performed with a fluorochrome-conjugated antibody directed against the primary antibody, e.g., Anti-IgG-FITC in case of a primary IgG-isotype antibody, or Anti-Biotin-PE in case of a biotinylated primary antibody. Of course, the fluorochrome-staining reagent itself has to be applied at its ideal concentration. The cells are then washed, suspended in flow buffer, and analyzed by flow cytometry.
