Cd19 clone 6d5

Lung ILC2 cell identification was performed as described previously (47 (link)). Lung tissues were digested in 8 mL RPMI 1640 containing liberase (50 μg/mL) and DNase I (1 μg/mL) for approximately 40 minutes at 37°C. Cell suspensions were filtered through 70 μm cell strainers and washed once with RPMI 1640. For ILC2 cell identification, total lung cell suspensions were blocked with 2.4G2 antibodies and stained with lineage cocktail mAbs: CD3ε (clone 145-2C11) (BioLegend, 100304), CD4 (clone GK1.5) (BioLegend, 100404), CD8α (clone 53-6.7) (Tonbo Biosciences, 30-0081-U500), CD11c (clone N418) (BioLegend, 117304), FceRIα (clone MAR-1) (BioLegend, 134304), NK1.1 (clone PK136) (BioLegend, 108704), CD19 (clone 6D5) (BioLegend, 115504), TER119 (clone TER-119) (BioLegend, 116204), CD5 (clone 53-7.3) (BioLegend, 100604), F4/80 (clone BM8.1) (Tonbo Biosciences, 30-4801-U500), Ly6G (clone RB6-8C5) (Tonbo Biosciences, 30-5931-U500), APC-conjugated streptavidin (BioLegend, 405207), PE-conjugated T1/ST2 (clone DIH9) (BioLegend, 145304), PerCP-Cy5.5-conjugated CD25 (clone PC61) (BioLegend, 102030), V450-conjugated Sca-1 (clone D7) (BD Biosciences, 560653), PE-Cy7-conjugated KLRG1 (clone 2F1/KLRG1) (BioLegend, 138416), APC-Cy7-conjugated CD45, and Fixable Viability Dye eFluor 506.

Single cell suspensions were stained with Fc receptor block (TruStain fcX, Biolegend) along with the appropriate anti-mouse antibodies: CD45 (AF700; clone 30-F11, Biolegend), TNFα (AF700; clone MP6-XT22, Biolegend), CD11b (BV605 or BV650; clone M1/70, Biolegend), Ly6C (BV711; clone HK1.4; Biolegend), Ly6G (APC-Cy7; clone 1A8), CD115 (APC; clone AFS98, Biolegend) or PE-Cy7; AFS98, eBioscience), CD68 (PE; clone FA-11, Biolegend). A lineage gate was used to exclude other cell populations and consisted of the following biotinylated antibodies: CD3 (clone 145-2C11, Biolegend), CD19 (clone 6D5, Biolegend), NK1.1 (clone PK136, Biolegend), Ter119 (clone TER-119, Biolegend), and SiglecF (PE-CF594; E50-2440, BD Biosciences). A fixable stain (LIVE/DEAD Blue, Life Technologies) was used to exclude dead cells. For intracellular staining, cells were first stained for surface markers, fixed in 2% PFA, then permeabilized in 0.5% Saponin (Sigma), and stained for intracellular markers. Fluorescence-minus-one controls were used to validate results. Samples were acquired on a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software v10 (FlowJo LLC, USA).

We first incubated cells with PE-conjugated anti-mouse antibodies directed against CD11b (clone M1/70), CD19 (clone 6D5), CD90.2 (clone 53-2.1), CD11c (clone N418), CD4 (clone GK1.5), CD8a (clone 53-6.7), CD127 (clone A7R34), CD49b (clone DX5), Ly-6G (clone 1A8), Ly-6C (clone HK1.4), TER-119 (clone TER-119) (all BioLegend). Then cells were stained with antibodies directed against c-kit (BioLegend, clone 2B8), sca-1 (BioLegend, clone D7), CD34 (BD Bioscience, clone RAM34), CD16/32 (BioLegend, clone 93), CD115 (eBioscience, clone AFS98). The term LSK refers to hematopoietic stem and progenitor cells based on their characteristic surface expression pattern (Lineage ^neg, Sca-1⁺, c-Kit⁺) [42 (link)], specified as Lin (CD11b, CD19, CD90.2, CD11c, CD4, CD8a, CD127, CD49b, Ly-6G, Ly-6C, TER-119) ^low, sca-1 ^high, c-kit ^high and was used throughout the manuscript as it best describes the cell population investigated. Granulocyte–macrophage precursors (GMP) were defined as Lin (CD11b, CD19, CD90.2, CD11c, CD4, CD8a, CD127, CD49b, Ly-6G, Ly-6C, TER-119) ^low, c-kit ^high, sca-1 ^low, (CD34/CD16/32) ^high, CD115 ^int/low. Monocyte-dendritic cell precursor (MDP) were defined as Lin (CD11b, CD19, CD90.2, CD11c, CD4, CD8a, CD127, CD49b, Ly-6G, Ly-6C, TER-119) ^low, c-kit ^int/high, sca-1 ^low, (CD34/CD16/32) ^high, CD115 ^high [25 (link)].

Antibodies directed against murine CD3 (clone 145-2C11, Biolegend), CD4 (clone RM4-5, Biolegend), CD8 (clone 53-6.7, Biolegend), NK1.1 (clone PK136, Biolegend), CD62L (clone MEL-14, Biolegend), CD44 (clone IM7, Biolegend), and CD19 (clone 6D5, Biolegend) were used in multicolor FACS analysis. Samples were washed and resuspended in cold flow cytometry staining buffer (1% BSA/PBS); stained for 30 min in the dark before a final wash and acquisition. All samples were acquired on a BD Fortessa Flow cytometer running FACS DIVA software. Analysis was performed using FlowJo X software (TreeStar; OR, United States).

The following directly conjugated antibodies were utilized for flow cytometry analysis: CD3 (clone 145-2C11; eBioscience), CD4 (clone GK1.5; Biolegend), CD8a (clone 53-6.7; BD Biosciences), CD11b (clone M1/70; Biolegend), CD11c (clone N418; Biolegend), CD19 (clone 6D5; Biolegend), F4/80 (clone BM8; Biolegend), and GR-1 (Ly-6G) (clone RB6-8C5; eBioscience). All antibodies were titrated on normal B6 splenocytes prior to use. Lung cells were prepared as above. After preparation of single cell suspensions, cells were blocked for 30 min at 4°C with 24G2 serum to block Fc receptors. Cells were then stained with indicated antibodies for 30 min at 4°C in the dark. Cells were then washed three times with PBS + 2%BSA and analyzed on a Becton Dickinson LSRII flow cytometer. FlowJo (Treestar) was used for all flow cytometry analysis.