Allophycocyanin apc conjugated anti human cd4

Samples of peripheral blood were collected by retro-orbital bleeding under general anesthesia and stained for 30 min with BV711-conjugated antihuman CD3, allophycocyanin (APC)-conjugated anti-human CD4, BB515-conjugated anti-human CD8, and BUV395-conjugated anti-human CD45 (all from BD Biosciences, San Jose, CA). Stained peripheral blood samples were then lysed with red blood cell lysis buffer, and absolute cell counts were calculated using BD liquid counting beads (BD Biosciences). Flow cytometry was performed using a BD Fortessa II instrument (BD Biosciences) and analyzed with FlowJo software.

WJ-MSCs were plated into 6-well plates (1 × 10⁵/well) and were incubated for 24 h with serum-free medium. Human PBMCs from healthy donor were cultured on top of the MSCs (MSCs/PBMCs ratio, 1:10) in the presence of 1 × 10⁵ Dynabeads Human T-Activator CD3/CD28 (Gibco, Billings, MT, USA) and 30 ng/mL human interleukin-2 (IL-2; PeproTech, Cranbury, NJ, USA). After 3 d of co-culture, the PBMCs were removed and analyzed by flow cytometry. PBMCs were stained with the following antibodies: FITC-conjugated FOXP3 monoclonal antibody (Thermo Fisher Scientific, Agawam, MA, USA), PE-conjugated anti-human CD25 (BD, San Jose, CA, USA), and allophycocyanin (APC)-conjugated anti-human CD4 (BD, San Jose, CA, USA), according to the manufacturer’s instructions. Samples were run on a FACSCanto™ flow cytometer (BD, San Jose, CA, USA). The percentage of proliferating T cells was analyzed by FlowJo version 10. For the PD-L1 blocking assay, 7.5 μg/mL PD-L1 neutralizing antibody (BPS Bioscience, San Diego, CA, USA) was added to the medium with the MSCs during 24 h. The antibodies were removed and washed-off with PBS before the addition of the PBMCs.