Flow cytometry is used to analyze membrane surface antigens and intracellular antigens expressed by cells, and is capable of specifically distinguishing different cell types in heterogeneous populations, evaluating the purity of isolated cell subpopulations, and analyzing cell size and volume. The technique primarily detects the fluorescence intensity of fluorescently coupled antibodies or ligands that bind a particular cellular antigen molecule.
Author: J.E. Colligan et al, Translator: Xuitao Cao et al, This experiment is from the "Compendium of Immunology Laboratory Guide".
Operation method
Immunofluorescence and cell separation Move Basic Scheme 1 Immunofluorescent labeling of single-cell surface antigens Materials 6 . Calculate the fluorescein/protein ratio (F/P ). Follow the FITC coupling method (see Supplementary Scheme 1), substituting the following reagents and steps as described in the Special Instructions. V Succinimidyl ethyl ester labeling buffer For more product details, please visit Aladdin Scientific website.
Basic Scheme 2 Immunofluorescent labeling of intracellular antigens in fixed and permeabilized single cells Materials 
Supplementary Option 1 Antibody coupled with fluorescent isothiocyanate yellow (FITC) Material 
F/P = F I T C moles/protein moles; F/P = 5 : 1 to 6 : 1 is most suitable for flow cytometry.
Additional material (see Additional Program 1 for additional material and Appendix 1 for items with V)
10m g /m l long-armed Biotin (Z y m e d Company) was dissolved in D M S O and prepared before use.
1 . Dialyze 1~2m g/m l of purified antibody in the same way as for FITC coupling. At the time of dialyzing, the antibody was prepared with a succinimide
Replace the FIC Labeling Buffer with Succinimidyl Esters Labeling Buffer. Determine antibody concentration by measuring absorbance at A 28.
(= A 280 X 0.74 X dilution).
2 . Add IOm I of lOmg/m l of long arm biotin prepared in anhydrous DMOS per mg of antibody. Incubate at room temperature for lh.
Remove unbound biotin in the same manner as for FITC (see Supporting Scheme 1).
Option 2 Labeling of dead cells with 7-aminoactinomycin D (7-A A D ) Additional material 