Cd4 apc cy7

50 µL whole blood was stained with cocktails of fluorochrome-conjugated surface antibodies to analyze for CD4 (CD4 APC-Cy7 or PerCP) and CD8 (CD8 Pacific blue) T cells expressing markers of Tregs (CD127 PE, FOXP3 APC); memory (CD45RO APC, CD62L PE-Cy7); terminal differentiation (CD57 FITC); activation (HLADR PerCP and CD38 PE-Cy7), proliferation (Ki67 FITC), and perforin production (Perforin PE) [Becton-Dickinson (BD) for all fluorochromes except for CD8 PB, FOXP3 APC, and CD62L PE-Cy7, which were from E-biosciences]. Red blood cells were lysed, and cells washed and incubated with surface antibodies for 30 min at 4°C. Cells for Treg analysis were then washed in 200 µL permeabilization buffer (E-biosciences); incubated with normal rat serum (1:50 dilution) for 15 min, 4°C in the dark, then FOXP3 added and incubated for 30 min at 4°C. Cells for Ki67 and perforin intracellular staining were washed with BD permeabilization buffer, Ki67 and perforin fluorochromes were added, and cells incubated for 30 min at 4°C in the dark. Stained cells were resuspended in 150 µL fix buffer (1% formalin in PBS), then acquired on the Cyan Flow cytometer (CyanADP). Flow cytometry data were analyzed using FlowJo software (Treestar, CA, USA) according to the gating strategies described (Figure 2).

1 × 10⁶ splenocytes were taken for antibody staining and downstream analysis. Specific monoclonal antibodies were purchased from eBioscience (Thermofisher Scientific, Waltham, MA, USA) or BD Biosciences (San Jose, CA, USA): CD4 APC-Cy7 (RM4-5), CD8 BV510 (53-6.7), CD11b BV510 (M170), CD11c PECy7 (HL3), CD19 PerCP-Cy5.5 (ID3), CD69 BV421 (H1.2F3), CD279 PE (PD1; J43), Gr-1 Alexa700 (Ly6G/Ly6C; RB6-8C5), MHC-II FITC (I-A/I-E; 2G9) and CD16/32 (2.4G2). H-2D^b restricted LCMV tetramer staining and peptide restimulation were performed as previously described [20 (link)]. All flow cytometry data were collected on a Fortessa X20 or Fortessa1 (BD Biosciences) and analyzed using FlowJo software (version 10, Flowjo LLC, Ashland, OR, USA).

To analyze the magnitude and phenotype of the HIV-1-, FLAG- or VACV-specific T cell immune responses, 2 × 10⁶ splenocytes (erythrocyte-depleted) seeded on 96-well plates were stimulated for 6 h in complete Roswell Park Memorial Institute (RPMI) 1640 medium (100 units/mL of penicillin/100 μg/mL of streptomycin, 2 mM L-glutamine, 10 mM Hepes and 0.01 mM β-mercaptoethanol) with 10% FCS, anti-CD107a-FITC (BD Biosciences), 1 µL/mL Golgiplug (BD Biosciences), monensin 1X (Invitrogen) and 5 µg/mL of the different HIV-1 clade B consensus peptide pools or 5 µg/mL of FLAG peptide or 10 µg/mL of VACV E3 peptide. After stimulation, splenocytes from immunized mice were stained for surface markers, fixed/permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and stained intracellularly with the following fluorochrome-conjugated antibodies: IL-2-APC, IFN-γ-PeCy7 and TNF-α-PE for functional analyses and CD3-PECF594, CD4-APCCy7, CD8-V500, CD127-PerCPCy5.5 and CD62L-Alexa700 for phenotypic analyses (all from BD Biosciences). The dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen).

Freshly thawed PBMCs were resuspended in serum-free, antibiotic-free RPMI 1640 media. Cells were distributed (0.5 x 10⁶cells in 100 μL per tube) to Falcon polystyrene round-bottom tubes (12 x 75 mm; Corning Incorporated, Durham, NC) and treated with IL-6 or IFN-γ (100 ng/mL) (R&D Systems, Minneapolis, MN) for 15 min at 37°C before subjecting them to phospho-specific flow cytometric analysis as described previously [7 (link)]. Briefly, after stimulation, cells were fixed by incubating in 2% PFA (BD Cytofix Fixation Buffer; BD Biosciences) for 10 min at 37°C and pelleted. They were then permeabilized by resuspending with vigorous vortexing in 300 μL ice-cold methanol. Cells were washed in staining media. Fluorophore-specific MAbs were added and incubated for 30 min at RT. The following markers were analyzed: CD3-PE/Cy7, CD4-APC/Cy7, CD45RO-PerCP/Cy5.5, CD33-APC, STAT1 (pY701)-AF488, STAT3 (pY705)-AF488, (BD Biosciences, San Diego, CA); and CD45-PE (R&D Systems, Minneapolis, MN). The cells were washed with staining media and pelleted. Finally, the samples were resuspended in 250 μL of staining media and analyzed.