Fitc conjugated anti cd19 antibody clone hib19

Along with the apoptosis assessment, the expression of CB2R was evaluated. Briefly, following the overnight exposure to the CM, 50 × 10³ cells were collected, washed in PBS, and stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD19 antibody (Clone HIB19; BD Biosciences, Franklin Lakes, NJ, USA) and an Alexa Fluor^® 647-conjugated Human Cannabinoid R2/CB2/CNR2 Antibody (Clone 352110R; R&D system). Data were acquired using the BD Accuri™ C6 flow cytometer (BD Biosciences) and analyzed using FCS Express™ 6 software (De NovoSoftware, Los Angeles, CA, USA). Our gating strategy was as follows: first, all CD19+ cells were identified in a dot plot, and then CB2R+ cells were identified in this population and the number of events enumerated.

In addition to cytotoxicity, PBMC apoptosis was assessed by using flow cytometry employing an Allophycocyanin (APC) conjugated-Annexin V Apoptosis Detection Kit (Cat# 640932; BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. Furthermore, the number of CD45+/CD19+ cells was measured in each experimental condition of the dose-response assay. Briefly, following the overnight exposure to the CM, 50 × 10³ cells were collected, washed in PBS, and stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD19 antibody (Clone HIB19; BD Biosciences, Franklin Lakes, NJ, USA) and an APC-conjugated anti-CD45 antibody (Clone HI20; BD Biosciences). Data were acquired using a BD Accuri™ C6 flow cytometer (BD Biosciences) and analyzed using FCS Express™ 6 software (De NovoSoftware, Los Angeles, CA, USA). Our gating strategy was as follows: first, all CD45+ cells were identified in a dot plot, and then CD19+ cells were identified in this population and the number of events enumerated. The acquisition volume was 200 µL.