Anti siglec f pe cf594

Dissociated lung cells were resuspended in 100 μL staining buffer and blocked with anti-CD16/32 antibody (BioLegend) followed by staining using fluorescently-tagged antibodies: anti-CD45 BV605 (BioLegend), anti-CD3 PE-Cy7 (BioLegend), anti-CD11b (BioLegend), anti-IA/IE BV650 (BioLegend), anti-CD11c (BioLegend), anti-CD24 AF488 (BioLegend), anti-CD193 PE (BioLegend), anti-GR1 AF700 (BioLegend), anti-CD64 APC (BioLegend), anti-Siglec-F PE-CF594 (BD Bioscience), along with a viability indicator Ghost Dye Violet 510 (Tonbo Bioscience). The list of antibodies, sources, and identifier (Cat#) information is summarized in the key resources table. The data acquisition was performed in LSRII Fortessa by collecting a total of 100,000 events (∼90% single cell events/samples) and analyzed using FCS Express 7 software. Dead cells were excluded based on Ghost dye⁺ (dead cells) and Ghost dye¯ live cells were included for further gating. Compensation for each flow cytometer experiment was performed with unstained and all single-color controls using OneComp eBeads (Thermo Fisher Scientific). Representative dot plots of a multicolor flow cytometry panel used to identify myeloid cell subsets in lung tissues from mice were provided as described (Figure S2).⁶⁰ (link)

Cells in the BAL compartment were incubated in human gamma globulin to block Fc receptors and prevent nonspecific binding of antibodies. Cells were washed and incubated in anti-CD3ε-Brilliant Violet 510 (BioLegend, San Diego, CA), anti-CD4-PE-Cy5 (BioLegend), anti-CD193-Alexa Fluor 647 (BD Biosciences, San Jose, CA), anti-Ly6G-V450 (BD Biosciences), and anti-Siglec-F-PE-CF594 (BD Biosciences) on ice for 30 minutes. Controls included unstained cells, isotype controls, and single color controls. Cells were washed and fixed with stabilizing fixative (BD Biosciences) and data were acquired with a BD LSR Fortessa and analyzed with FlowJo v10.1r5 (Ashland, OR).

Inguinal lymph nodes were harvested and processed to obtain single cell suspension. Total LN cells were then stained with anti-CD16/32 antibodies for blocking of Fc receptors, followed by staining with biotinylated anti-CD3 (Biolegend #100244) and anti-CD19 (eBioscience #13-0193-82) antibodies for 20 min at 4 °C. Next, samples were incubated with streptavidin-conjugated magnetic beads (BD #557812) for 30 min at 4 °C. and passed through a magnet (STEMCELL technologies), according to the manufacturer’s protocol. The negative fraction was transferred into a clean tube and stained for FACS sorting. Antibodies used for sorting include Live/Dead Fixable Aqua (BV510; Tonbo Biosciences #13-0870-T100), anti-CD19 (APC; Biolegend #152410), anti-CD3 (APC; Biolegend #100236), anti-CD45 (BV610; Biolegend #103140), anti-CD11c (BV421; Biolegend #117330), anti-CD11b (FITC; Biolegend #101206), anti-Ly6C (BV780; Biolegend #128041), anti-Ly6G (APC-Cy7; Biolegend #127624), anti-Siglec-F (PE-CF594; BD #562757), anti-PDCA-1 (PE; Biolegend 127010). Samples were sorted on a FACSAria Fusion instrument.