Cd62l pe cy7

Multi-parameter flow cytometry was performed in accordance with the BD Pharmingen^TM protocol. In brief, mouse lungs were harvested at day 14 post second boost; lung lymphocytes (5x10⁶) were stimulated in Nunc^TM 96-well conical bottom plates (Thermo Scientific™, USA) for 6 h at 37°C with 10 μg of M2eh peptide, in combination with 1 μg of either the A/Aichi/2/68 (H3N2) or the A/California/07/09 (H1N1pdm09) influenza virus, in the presence of 1 μg/ml of Brefeldin A (BD Bioscience, USA) and purified hamster anti-mouse CD28. The cells were then washed, and Fc receptors were blocked using CD16/CD32 antibodies (Mouse BD Fc Block, BD Pharmingen, USA) for 30 min. Next, cells were incubated with Zombie Aqua (Zombie Aqua™ Fixable Viability Kit, Biolegend, USA) to enable gating of live cells during analysis, and they were subsequently stained with CD3e-FITC, CD8a- APC-Cy™7, CD4- PerCP, CD62L-PE-Cy™7, or CD44-APC (BD Pharmingen, USA) antibodies at 4⁰ C for 30 min. Cells were permeabilized using the BD Cytofix/Cytoperm Plus (BD Bioscience, USA) protocols and stained with anti-TNF-α-BV421 or anti-IFN-γ-PE antibodies (BD Pharmingen, USA). Sample acquisition (50,000 live CD3+ T-cells were collected) was performed with a BD FACS Canto II flow cytometer (Becton Dickinson, USA) and analyzed using Kaluza version 1.5 (Beckman Coulter, USA).

Hepatic mononuclear cells (HMNCs) and splenocytes were isolated as previously described [18 (link)]. Single-cell suspensions of HMNCs and splenocytes were labelled with fluorescent conjugated antibodies against mouse CD4-FITC, CD25-PE (eBioscience), CD62L-PE Cy7, CTLA-4-PE (BD Pharmingen), as well as CD103-PerCP-Cy5.5 (Biolegend). Nonspecific binding was blocked with staining buffer supplemented with 2% mouse serum and anti-CD16/anti-CD32. For intracellular staining of Foxp3, mouse regulatory T cell staining Kit (eBioscience) was used. Cells were labeled with surface antibody, fixed and permeabilized with freshly prepared Fixation/Permeabilization working solution according to the manufacturer’s instructions. After permeabilization, cells were further labeled with anti-mouse foxp3 (clone FJK-16s) (eBioscience). After incubation and resuspension, HMNCs and splenocytes were evaluated by flow cytometry, and the data were analyzed using Cell Quest software (Becton Dickinson).

Washed with FACS buffer (PBS with 2% FBS), prepared lung single cells were incubated with unlabelled anti-CD16/CD32 (eBioscience) to block the Fc receptors. After washed and suspended by the FACS buffer, blocked cells were stained with anti-CD45-PerCP (BD Biosciences), anti-CD11b-FITC (BioLegend), anti-Ly-6G-PE (Sungene), anti-CD64-APC (BioLegend), and CD62L-PE-Cy7 (BD Biosciences) for 30 min in the dark at 4°C to indicate the pulmonary neutrophils. Then, the cells were fixed with 2% paraformaldehyde (PFA) in PBS for 30 min at 4°C, and 100 μl FACS buffer was finally added to resuspend the fixed cells. The flow cytometry analysis was conducted on FACS Canto II flow cytometer (BD Biosciences), and acquired data were analyzed using the Flow Jo software version 10 with pulmonary neutrophil subsets being defined as CD45⁺ CD11b⁺ Ly-6G⁺ cell.

Thawed PBMCs were reacted with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA) to remove the dead cells. For phenotypic analysis, cells were stained with phycoerythrin (PE)-conjugated Tax_301-309/HLA-A*24:02 tetramer reagents (MBL, Nagoya, Japan) and several fluorescence-conjugate mouse anti-human monoclonal antibodies (mAbs) [CD3-APC-H7, CD8-Pacific Blue, CD45RA-PerCP5.5, CCR7-Alexa647, CD62L-PE-Cy7, CD27-FITC, CXCR3-BV605 (BD Biosciences), and CD95-PE-Cy5 (Biolegend, San Jose, CA)] for 25 min on ice. Stained cells were washed twice and immediately acquired using FACSAriaIII Fusion (BD Biosciences) equipped with 20 detectors by 4-lasers at 488 nm, 561 nm, 633 nm, and 405 nm. The data were analyzed using FlowJo ver.10 software (BD Biosciences). The experiments requiring cell sorting for TCR repertoire analysis, described below, were carried out using the same equipment.

We purchased directly conjugated monoclonal antibodies for flow cytometry from Invitrogen (Waltham, MA, USA): CD4-PE, Clone GK1.5; CD11b-ef450, Clone M1/70; CD44-FITC, Clone IM7; CD45AF700, Clone 30-F11; CD206-APC, Clone MR6F3; FoxP3-ef450, Clone FJK-16S, IFN-γ-PE, Clone XMG1.2; GzmB-APC, Clone GB11; iNOS-PE, Clone CXNFT; live dead fixable dead cell stain kit, Catalogue No. L34959. BD Pharmingen (San Jose, CA, USA): CD3-AF700, Clone 17A2; CD4-Pacblue and perCPcy5.5, Clone RM4-5; CD8-FITC, perCPcy5.5, and PECY7, Clone 53-6.7; CD62L-PECY7, Clone MEL-14; H2Kd-PE, Clone 17A2; Ly6G-PE and percpcy5.5, Clone 1A8; Ly6C-APCCY7, Clone- AL-21; TNF-α PECY7, Clone MP6-XT22. Biolegend (San Diego, CA, USA): CD3-PECY7 Clone 17A2; B220-percpcy5.5, Clone RA3-6B2. R&D Systems: CCR2-APC, Clone 475301; CCL2-APC, Clone 123616. eBioscience (San Diego, CA, USA): H2Kd/Dd-ef450 Clone 34-1-25; CD4-APC, Clone GK1.5.

After incubation at 37°C, 5% CO₂, cells were incubated at 4°C for 15 minutes, stained with fluorochrome conjugated monoclonal antibodies, and suspended in saline. Antibodies were: CD16 PE, CD62L-PECy7 and CD66b-PerCPCy5.5 (BD Pharmingen, San Diego, CA, USA). Flow cytometry data (at least 50,000 events per sample) were acquired using either a FACSVerse Flowcytometer (BD Bioscience) or a FACS CantoII (BD Bioscience). Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).
Neutrophils were identified by forward- and side-scatter characteristics and, in some cases, CD16+ expression. Infected or bystander population of neutrophils were identified as CFSE+ or CFSE- cells, respectively (S1 Fig).
As the experiments with healthy controls subjects were performed at the laboratory at Complexo Hospitalar Universitário Professor Edgard Santos in Salvador city, we used a different flow cytometry than we have at the laboratory in the endemic area.