Following are two protocols for the detection of cell surface antigen by flow cytometry using antibodies: a procedure for staining with directly-labeled antibodies and another for labeling with unconjugated antibodies followed by a fluorochrome-labeled secondary antibody.

Technical Considerations:

  • Fluorochrome selection: See our fluorochrome table for commonly utilized fluorochromes and consult Core Facility personnel regarding available laser excitation wavelengths. When combining multiple antibodies, consider fluorochrome emission overlap.
  • Obtaining a single cell suspension: If working with cells from a monolayer, exercise care in choice of removal medium, as some enzymatic methods may digest the epitope of interest.
  • Cell Viability: Cells should be in viable condition upon staining with antibody. Dead cells will stain with antibody non-specifically and also exhibit high autofluorescence. Viability can be ascertained either prior to staining by microscopy (trypan blue dye exclusion) or upon flow cytometry acquisition (unfixed cells only) using membrane impermeant DNA dyes (Propidium Iodide, 7-aminoactinomycin D, etc.). More info
  • Use of blocking serum: To reduce background fluorescence due to non-specific antibody binding, we recommend resuspending cells to be stained in PBS containing serum. For indirect labeling procedures, use minimum of 10% (and up to 50% depending on severity of problem) serum from the species of the secondary antibody. For directly-labeled antibodies the serum source should be that of the antibody species. Alternately, you can use a PBS buffer with 2% BSA.
  • Recommended controls for Immunophenotypic analysis:
    • Unstained cells: to determine background autofluorescence
    • Secondary antibody only: assess background fluorescence due to the use of secondary antibody alone
    • Primary antibody only: same as above
    • Irrelevant immunoglobulin (isotype) control: should be of the same species, isotype and concentration as the test antibody, but directed against an irrelevant epitope
    • Positive control: known biological positive test sample
    • Negative control: known biological negative test sample
  • Antibody titration: The antibodies may arrive with a target concentration recommended by the manufacturer. It is recommended that a minimum five-point titration be performed to optimize the signal to noise ratio of the antibody staining. When titering an unconjugated antibody, use twice the amount of secondary antibody at each point of the titer.
  • Cell number: Number of cells per test should be consistent, and should not exceed 1×106.

Antibody Labelling – Direct Protocol
Antibody Labelling – Indirect Protocol
Assessment of Cell Viability