Percp cy5.5 anti cd19

Fasting venous blood samples were collected from individual subjects, and peripheral blood mononuclear cells were isolated by density‐gradient centrifugation using Ficoll‐Paque Plus (Amersham Biosciences, Little Chalfont, UK). Peripheral blood mononuclear cells at 1 × 10⁶/tube were stained in duplicate with APC‐H7‐anti‐CD3, PerCP‐Cy5.5‐anti‐CD19, PE‐Cy7‐anti‐CD27, APC‐anti‐CD38, PE‐anti‐CD138, PE‐CF594‐anti‐CD20, FITC‐anti‐IgD (BD Biosciences, San Jose, CA, USA) in the dark at room temperature for 30 min. After being washed, the cells were fixed and permeabilized using a fixation/permeabilization kit (BD Biosciences), followed by intracellular staining with BV510‐anti‐IgG, and BV421‐anti‐IgM (BD Biosciences). Negative controls were stained with isotype‐matched control antibodies (APC‐H7‐anti‐IgG1, PerCP‐Cy5.5‐anti‐IgG1, PE‐Cy7‐anti‐IgG1, APC‐anti‐IgG1, PE‐anti‐IgG1, PE‐CF594‐anti‐IgG2b, FITC‐anti‐IgG2a, BV421‐anti‐IgG1, BV510‐anti‐IgG1; BD Biosciences). The percentages of different subsets of B cells were characterized on a FACSAria II (Becton Dickinson, San Jose, CA, USA) and at least 50,000 events were analysed by FlowJo software (v5.7.2; TreeStar Inc., Ashland, OR, USA). The number of each type of cells tested was calculated, according to the percentages of this type of cells multiplied lymphocyte counts.

Isolated PBMCs were stained in duplicate with FITC-anti-CD3 (BD Biosciences, San Diego, CA, USA), PerCP-Cy5.5-anti-CD19 (BD Biosciences), BV421-anti-CD161 (BD Biosciences), APC-anti-TCRVα7.2 (BioLegend, San Diego, CA, USA), PE-CF594-anti-TCRγδ (BD Biosciences), PE-Cy7-anti-CD8α (BD Biosciences), and PE-anti-CD8β (BD Biosciences) antibodies at 4 °C for 30 min in the dark. Isotype-matched control antibodies were used as negative controls. The frequencies of various T-cell subsets were determined by flow cytometry analysis using the FACSAria II (BD Biosciences) and FlowJo software (v7.6.2; TreeStar, San Carlos, CA, USA).

Primary mouse peripheral blood mononuclear cells (PBMCs) were isolated from mouse blood through density gradient centrifugation according to the manufacturer’s instructions (Solarbio, Beijing, China) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. PBMCs were treated with curcumol (0, 25, 50, 100 μM) for 36 h, and single-cell suspensions were incubated and stained with PE-anti-CD4 (Biolegend, CA, USA), FITC-anti-CD8a (BD Biosciences, NJ, USA) and PerCP/Cy5.5-anti-CD19 (BD Biosciences, NJ, USA) antibodies for 30 min at room temperature. Then cells were washed and the percentages of different immune cells in PBMCs by flow cytometry were analyzed.