Pe conjugated anti cd28

Samples of 100 μL per tube blood were transferred for staining with monoclonal antibodies and red blood cell (RBC) lysis. RBCs were lysed with buffer containing 0.8% NH₄Cl and 0.1% KHCO₃. Cells were then washed with PBS (phosphate-buffered saline) and stained with FITC-conjugated anti-CD3 or anti-CD95, RPE-Cy5-conjugated anti-CD4 (BD Pharmingen, San Diego, CA, USA), APC-H7-conjugated anti-CD8, PE-conjugated anti-CD28, anti-CD25, anti-CD69, or anti-HLA-DR (BD Pharmingen, USA) for 30 min at 4 °C in the dark. Then, cells were washed with PBS and suspended in 200 mL of suitable buffer for flow cytometric analysis using a FACSVerse instrument (Becton Dickinson, Franklin Lakes, NJ, USA).

PBMCs were isolated by centrifugation on Histopaque™ gradient (Sigma Chemical Co., USA) from venous peripheral blood collected in tubes containing EDTA as the anti-coagulant. Cells were then stained with 3 µM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, Sigma Chemical Co., USA), stimulated with 250 ng per 2 million cells of immobilized (tissue-culture plate-bound) anti-CD3 antibody (BD-Pharmigen, USA) and incubated for up to 5 days at 37 °C, 5% CO₂ as previously described^{11 (link)}.
Stimulated cells were collected after 72 and 120 hours, stained with following antibodies: RPE-Cy5-conjugated anti-CD4 or anti-CD8 and PE-conjugated anti-CD28 (BD Pharmingen, USA), and analyzed with flow cytometry using FACScan instrument.

Flow cytometric analysis was performed using blood samples collected before treatment (weeks 7–8 post-immunization) and at 2, 3, 5, 6, 7, and 8 weeks after treatment (weeks 9–10, 10–11, 13, 14, 15, and 16 post-immunization). All antibodies were purchased from BD Biosciences (San Jose, CA, USA). For analysis of T-cell populations, peripheral blood mononuclear cells (PBMCs) were stained with V500–conjugated anti-CD45 (BD Biosciences), PE-Cy7–conjugated anti-CD3, PerCP-Cy5.5–conjugated anti-CD4, APC-Cy7–conjugated anti-CD8, PE–conjugated anti-CD28, and APC–conjugated anti-CD95 antibodies. For analysis of B-cell populations, PBMCs were stained with V500–conjugated CD45, PE-Cy7–conjugated anti-CD3, FITC–conjugated anti-CD20, V450–conjugated anti-CD27, PerCP-Cy5.5–conjugated anti-CD21, PE–conjugated anti-IgD, and APC–conjugated anti-IgM antibodies. For analysis of regulatory T cells, PBMCs were stained with V500–conjugated anti-CD45, FITC–conjugated anti-CD4, PE-Cy7–conjugated anti-CD25, and PE–conjugated anti-FoxP3 antibodies. Flow cytometric analysis was carried out using a BD LSRFORTESSA cell analyzer (BD Biosciences).