Pe cy7 mouse igg2a

For the analysis of CD24 and CD44 expression on cell membrane, 5 × 10⁵ of cells were collected in Cell Staining Buffer (BioLegend, #420201) and stained with PE-Cy™7-conjugated anti-CD24 (1:100 for 20 min; BD Biosciences, #561646) and APC-conjugated anti-CD44 antibody (1:60 for 20 min; BD Biosciences, #559942) by using PE-Cy™7 Mouse IgG2a (1:100 for 20 min; BD Biosciences, #552868) and APC Mouse IgG2b (1:60 for 20 min; BD Biosciences, #555745) as control staining. Stained cells were analyzed by BD FACSCanto II cytometer and data were acquired by BD FACSDiva v8.0.2 software and processed by FlowJo v10.7.1 software (BD Biosciences). ALDEFLUOR assay was carried out using the ALDEFLUOR assay kit (STEMCELL Inc., #101700) according to the manufacturer’s instructions. The ALDH1 inhibitor, diethylaminobenzaldehyde (DEAB), was used as a negative control. The processed cells were evaluated by BD FACSCanto II cytometer and data were acquired by BD FACSDiva v8.0.2 software and analyzed by FlowJo v10.7.1 software (BD Biosciences).

PE mouse IgG1(BD), APC Rat IgG2a (BD), PerCp/Cy5.5 mouse IgG2b (Biolegend), Alexa Fluor 700 Mouse IgG1 (BD), PE-Cy7 Mouse IgG2a (BD), V450 Mouse IgG1 (BD), APC Cy7 Mouse IgG1 (Biolegend), FITC Mouse IgG2a (BD).
Following staining, cells were fixed with 2% paraformaldehyde and run on a BD Biosciences LSR II flow cytometer (San Jose, CA, USA). Data were analyzed using Winlist 6.0 (Topsham, ME, USA). Results are reported as the percent of the phenotype being analyzed in the total peripheral blood mononuclear cell pool.