Cd4 peridinin chlorophyll protein percp cy5.5 rm4 5

Erythrocytes were lysed using lytic lysis buffer (Sigma-Aldrich) as recommended by the manufacturer. Cells were pelleted at 400 × g, washed in phosphate-buffered saline containing 2% fetal bovine serum, and stained with the following antibodies: panel 1, CD3-BUV.395 (145-2C11; BD Biosciences), CD4-peridinin chlorophyll protein (PerCP).Cy5.5 (RM4-5; BD Biosciences), CD8-BVLT.421 (53-6.7; BD Biosciences), IFN-γ–Alexa.488 (XMG1.2; BD Biosciences), IL-17A–phycoerythrin (PE) (TC11-18H10.1; BD Biosciences), and IL-4–allophycocyanin (APC) (11B11; BD Biosciences); panel 2, CD11b-Alexa.488 (M1/70; BioLegend), Ly6G/Ly6C-Alexa.647 (RB6-8C5; BioLegend), CD64-BVLT.421 (X54-5/7.1; BioLegend), CD69-PE (H1.2F3; BioLegend), CD3-BUV.395, and CD4-PerCP.Cy5.5; panel 3, CD19-APC (1D3; BD Biosciences), CD14-BUV.737 (rmC5-3; BD Biosciences), and CD335-fluorescein isothiocyanate (FITC) (29A1.4; BD Biosciences). For determination of absolute counts, CountBright absolute counting beads (Thermo Fisher Scientific) were used per the manufacturer’s instructions. Data were collected using an LSRII Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo.

Peripheral blood was collected in tubes containing EDTA (Kent Scientific Corp.; catalog identifier MTSC-EDTA) at day 6 postchallenge. Erythrocytes were lysed using lysis buffer (Sigma-Aldrich) as recommended by the manufacturer. Cells were pelleted at 400 × g, washed in phosphate-buffered saline containing 2% fetal bovine serum (Thermo Fisher Scientific), and stained with the following antibodies: CD3-brilliant UV 395 (BUV395) (145-2C11; BD Biosciences), CD4-peridinin chlorophyll protein (PerCP)/Cy5.5 (RM4-5; BD Biosciences), IFN-γ–phycoerythrin (PE) (XMG1.2; BD Biosciences), TNF-α–fluorescein isothiocyanate (FITC) (MP6-XT22; BD Biosciences), and IL-2–allophycocyanin (APC) (JES6-5H4; BD Biosciences).