Cd19 apc

Monoclonal antibodies (CD3-FITC, CD19-APC, and annexin V-FITC) were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Appropriate APC, PE, and FITC isotype antibodies were used as negative controls (BD Biosciences). The fluorescent nanoparticle size standard kit was purchased from Spherotech (Lake Forest, IL, USA; Cat. No. NFPPS-52-4K). Elaiophylin was provided by North China Pharmaceutical Group Corporation New Drug R&D center. Stattic was purchased from Sigma-Aldrich.

PBMCs (up to 2x10⁷ cells) suspended in RPMI-1640 medium were labeled with 1 μL of 5 mM CFSE solution (Thermo Fisher Scientific, USA) and incubated in dark for 6 minutes at 4°C. Cells were washed twice with PBS and were counted under light microscopy with trypan exclusion method. CFSE-labeled cells were cultured in 48-well flat bottom culture plates (NEST Biotechnology, China) for 120 hours at 37°C in 5% CO₂ environment with the absence or existence of 5 µL/mL phytohemagglutinin (PHA, Thermo Fisher, USA). After the incubation, cell-culture supernatants were collected for future analysis and PBMCs were washed with PBS. After washing, PBMCs were re-suspended and 100 μL cell suspension was taken from the respective well (US: unstimulated, PHA: Phytohemagglutinin-stimulated) in 2 separate tubes for flow cytometry. Cells were labelled with anti-CD4-PE/Cy7 (BD Biosciences, USA) and -CD19-APC (BD Biosciences, USA) mAbs and incubated at dark for 20 minutes. After the incubation, cells were washed and re-suspended with FACS buffer. Samples were analyzed by a FACSCalibur flow cytometer running CellQuest software (BD Biosciences, San Jose, USA). The obtained data was evaluated as % proliferation. The proliferation percentages of total lymphocytes, CD4⁺ T cells and CD19⁺ B cells were evaluated.