Multicolor Flow Cytometry for Immune Cells

Cells were stained with the indicated monoclonal antibodies conjugated or not to fluorescein isothiocyanate (FITC), phycoerythrin (PE), or Cy-chrome 5 (Cy); anti-Human Fas Ligand (Fas-L), anti-human Vβ8 (BD Pharmingen) anti-human CD4 (Biocience), and/or dyes: Annexin-V (BD bioscience), Propidium Iodine (Sigma) 3,3`-diethyloxacarbocyanine iodine (DiOC₂ (3)), 5,6 carboxyfluorescein diacetate succinimidyl ester (CFSE), (Molecular Probes, Eugene, OR, USA). Briefly, cells (1 × 10⁶) were incubated with the appropriate antibody or dye for 20 minutes at room temperature in the dark (26 (link)) and washed with PBS. For Flow cytometry analysis, 30,000 events were collected in linear mode for forward scatter and side scatter and log amplification for FL-1, FL-2, and FL-3 using a FACScan cytometer (BD Biosciences). Background values were obtained with isotype controls (BD Pharmingen). Results were analyzed by using Cell Quest software (BD Immunocytometry System).