Percp conjugated anti cd4 antibody

Blood samples were obtained from healthy, HLA-A2-positive donors. Mononuclear cells were isolated using the Ficoll-Paque™ Plus (GE Healthcare Life Sciences, Marlborough, MA, USA) method. A total number of 1 × 10⁶ cells per well were plated on 24-well plates and incubated with the appropriate mRNA carrier containing 200 ng of each indicated mRNA per well (1 mL cultures). Starting day 4, every other day of the culture, 10 U/mL IL-2 (R&D Systems, Minneapolis, MN, USA) was added to the cells. Cells were cultured for 12 days. At day 12, cells were stained with phycoerythrin (PE)-HLA-A*0201 tetramers containing the conserved immunodominant HLA-A*0201 epitope from influenza matrix M1 [16 (link)], at a final concentration of 5 μg/mL in PBS (The Tetramer Shop, Kongens Lyngby, Denmark) during 15 min at room temperature. Then, the cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibody and a peridinin–chlorophyll–protein complex (PerCP)-conjugated anti-CD4 antibody (Becton Dickinson, Heidelberg, Germany) in a PBS buffer supplemented with 0.5% bovine serum albumin and 2 mM EDTA for 30 min at 4 °C. The cells were washed twice with PBS before being fixed with 1% formaldehyde and analyzed by flow cytometry (LSRForstessa, BD Biosciences, New Jersey, NJ, USA).

Upon activation, PBMCs were detached, washed, and stained with peridinin chlorophyll (PerCP)-conjugated anti-CD4 antibody (Becton Dickinson, San Diego, CA, USA) at room temperature for 30 min, fixed with 4% paraformaldehyde and permeabilized using PBS containing 0.5% saponin and 10% fetal bovine serum (FBS) at room temperature for 30 min. After three washes, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-IFN-γ, Alexa-Fluor647-conjugated anti-IL-17 (Becton Dickinson), and phycoerythrin (PE)-conjugated anti-IL-22 (R&D Systems, Minneapolis, MN, USA). Flow cytometry was performed on a FACS Calibur (Becton Dickinson) and analyzed using the FlowJo software (TreeStar, San Carlos, CA, USA).

Lymphocytes (0.5–1×10⁶) from primed cultures were stained with PerCP-conjugated anti-CD4 antibody (BD-Pharmingen) and PE-conjugated anti-Thy1.2 antibody (BD-Pharmingen), and cell division was analyzed by CFSE dilution. The absolute number of cell divisions accumulated within the CD4+ T cell subset was calculated as previously described [18] . BrdU incorporation by individual cells was detected using a Phoenix Red-conjugated anti-BrdU antibody (Phoenix Flow Systems) after fixation and permeabilization (Fix & Perm, Caltag Laboratories). DNA content was measured by the addition of 7-AAD (25 µg/ml) to the fixed and permeabilized cells for 30 min. The above fluorescence parameters were assessed using a FACSCalibur flow cytometer (Becton-Dickinson).