Flow Cytometric Analysis of T-Cell Subsets

Peripheral blood samples collected using blood collection tubes with spray-dried K2EDTA (BD, USA) were also used for flow cytometry. All biochemical testing reagents were purchased from BD Biosciences (USA). Peripheral blood lymphocyte subsets were analyzed on the BD FACSCanto Ⅱ Flow Cytometer (BD Biosciences). BD Multitest CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC reagent (BD Biosciences) containing anti-CD3 fluorescein isothiocyanate, anti-CD8 phycoerythrin, anti-CD45 peridinin chlorophyll protein, and anti-CD4 allophycocyanin was used for identifying and determining the percentages and absolute counts of mature human T lymphocytes (CD3⁺), suppressor/cytotoxic (CD3⁺CD8⁺) T lymphocyte subsets, and helper/inducer (CD3⁺CD4⁺) T lymphocyte subsets in erythrocyte-lysed whole blood (Fig. 1). Briefly, the reagent cocktail (10 μL) was added to 50 μL EDTA-anticoagulated whole blood, and the sample was mixed and incubated for 30 min at room temperature in the dark. Erythrocytes were lysed by adding 500 μL of ammonium chloride hemolysis agent (BD Pharm Lyse, USA) for 15 min. Then the cells were washed, incubated with 2% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4), and measured on the flow cytometer^{[9 (link)]}.