Cd45 percp cy5

Cells in BAL fluid were stained with CD3 FITC, CD11c PercP, Siglec F Alexa 647, CD11b PE-Cy7, viability dye APC Cy7 (all BD Biosciences, San Jose, CA, USA), Ly6G Alexa700 (Biolegend, San Diego, CA, USA), MHCII PE, and CD45 PE-eFluor610 (eBiosciences, San Diego, CA, USA) in the presence of Fc blocker (CD16/CD32, eBiosciences). Single cell suspensions from lungs were stained with CD4 FITC, CD45 PerCP-Cy5.5 (eBiosciences), GATA-3 Alexa 647, and viability dye APC-Cy7 (BD Biosciences). The following markers were used for the analysis of ILCs in lung tissue: Lineage (Lin) markers including CD3e, CD19, GR1, B220, Ter119, FcaR1 (all FITC, Biolegend), CD45 Alexa700, CD90 PE, ST2 Brilliant Violet 421 (Biolegend), CD49b PE-Cy7 (eBiosciences) and CD3 Percp-Cy5.5 (BD biosciences). Mediastinal lymph nodes (mLN) cells were stained with CD45 PerCP-Cy5.5, CD4 FITC, GATA-3 Alexa 647 (BD Biosciences) and IL-4 APC (Biolegend). For intracellular/intranuclear staining, cells were permeabilized and fixed using a FOXp3 Staining Buffer set (eBioscience) and subsequently stained with the appropriated markers. All appropriate Fluorescence Minus One (FMO) controls were used. Data were collected on a BD Biosciences Canto II flow cytometer or BD FACSAria™ III and analyzed using FlowJo software (Treestar, Palo Alto, CA, USA).

All cells were stained with Live/Dead Fixable Aqua (1:2000; Invitrogen) at room temperature for 30 min in the dark. Following the Live/Dead viability stain, cells were incubated with anti-mouse CD16/CD32 (Fc block; 1:100, eBioscience), CD3 PE-eFluor610 (1:100; eBioscience), CD4 PE (1:100; eBioscience), CD8b APC-eFluor780 (1:100; eBioscience), CD11b PE-Cy7 (1:200; eBioscience), Ly6G Pacific Blue (1:100; BioLegend), CD45 PerCP-Cy5.5 (1:100; eBioscience), CD11c Alexa Fluor 700 (1:50; BioLegend) Ly6C FITC (1:200; eBioscience) in FACS buffer. Samples were run on an LSRII (BD Biosciences) flow cytometer and analyzed with FlowJo_V10 software.
Single cell lymphocytes were gated on forward scatter area (FSA; size) and side scatter area (SSA; granularity) and then by forward scatter height (FSH) and FSA. Live cells were selected as the negative Fixable Aqua population. CD45⁺ cells were selected and gated by CD3 (T cells) versus CD11b (myeloid cells). The CD3⁺ population was then gated on CD4 (helper T cells) and CD8 (cytotoxic T cells). CD11b⁺ cells were further gated on CD11c and Ly6G for the following populations: CD11c⁻Ly6G⁻ (microglia and macrophages), CD11c-Ly6G⁺ (neutrophils), and CD11c⁺Ly6G⁻ (dendritic cells). CD11c⁻Ly6G⁻ cells were gated for CD45 and low and high populations were further gated for MHCII and Ly6C to determine their activation state (Suppl. Fig. S1).

The following antibodies were used to stain PCW cells: B220 PerCP-Cy5.5 (BioLegend, 103236), CD5 PE-Cy7 (BioLegend, 100622), CD19 BUV395 (BD Biosciences, 563557), IgM APC (Biolegend, 406509), PD-L2 BV421 (BD Biosciences, 564245), CD11b APC-Cy7 (BioLegend, 101226), Live/dead Ghost violet 510 (Tonbo Biosciences, 13-0870). For cells isolated from PZTD^+/+ mice, ZsGreen fluorescent protein was analyzed using the same channel as FITC. For cells isolated from CD19-Cre^+/- PTZD^+/+ mice, TdTomato fluorescent protein was analyzed using the same setting for TexasRed. To analyze TILs, CD45 PerCP-Cy5.5 (eBioscience, 45-0451-80), was added to the above panel replacing B220 for leukocyte gating. The stained samples were analyzed at the Boston University Medical Campus Flow Cytometry core facility using a BD LSRII flow cytometer and a Beckman Coulter MoFlo for cell sorting. Live leukocytes were gated by forward and side scatters, live/dead Ghost violet 510 and CD45 staining. B1 cells were gated as B220IntCD5Int population from the live leukocyte gate. L2pB1 cells were gated as PD-L2+IgM+ from the B1 cell gate. In the transgenic-knock in mice, L2pB1 cells were also identified by the expression of ZsGreen and TdTomato (Supplementary Figure S1).

Isolation of Intestinal lamina propria cells was performed by following a method established previously58 (link) with slight modifications. Small intestines were washed with three times with HBSS (Ca/Mg-free), and fat and Peyer's patches were removed. Small intestines were then opened longitudinally, cut into 1-cm pieces, and incubated in HBSS containing 5 uM EDTA+5%FBS+1μM DTT. Tissue was then digested 0.14 Wünsch U ml⁻¹ Liberase (Sigma) for 30 mins at 37 °C on a rotor. The digested cell suspension was then passed through 100 μm cell strainers. Isolated intestinal cells were stained with CD45 Percpcy5.5 (0.35 μl per 100 μl; #45–0451–82, eBioscience, San Diego, CA) CX3CR1 PE-TexasRed (1.5 μl per 100 μl; #149013, Biolegend, San Diego, CA) F4/80 AF647 (8 μl per 100 μl; #MCA497A647, Bio-Rad, Hercules, CA), CD11b eflour605 (2 μl per 100 μl; #83–0112–42, eBioscience) Ly6C PE/cy7 (1 μl per 100 μl; #128018, Biolegend) Ly6G APC/cy7 (0.3 μl per 100 μl; #127624, Biolegend) and were subjected to flowcytometric sorting to purify intestinal macrophages using FACS Aria machine (BD).