Anti mouse ly6g

Spleen and lymph nodes were harvested from immunized mice five days after the final boost. B cells were enriched using theEasySep mouse pan-B cell isolation kit (Stemcell Technologies, Vancouver, CA). Cells were then stained with a fluorophore-labeled antibody panel, which includes PerCP-Cy5.5 conjugated anti-mouse IgM (BD Biosciences), PerCP-Cy5.5 conjugated anti-mouse IgD (BD Biosciences), APC-Cy7 conjugated anti-mouse CD19 (BD Biosciences), and a cocktail of phycoerythrin (PE) conjugated antibodies used during sorting as a dump channel: anti-mouse Ly6g (Biolegend, San Diego, CA, USA), PE anti-mouse CD3 (Biolegend), and PE anti-mouse F4/80 (Biolegend) and Alexa647 and Alexa488 ovalbumin (InvivoGen, San Diego, CA, USA). Single dump channel-/CD19+/IgM-/ovalbumin^duel+-stained B cells were FACS sorted and barcoded using the Chromium controller (10× Genomics, Pleasanton, CA, USA) and a sequencing library was prepared according to the manufacturer’s instructions using the Chromium Next GEM single-cell 5′GEM, BCR amplification and library construction kit (10× Genomics, Pleasanton, CA, USA).

Five-micrometer thick sections were cut from formalin-fixed and paraffin-embedded tissue specimens and put onto slides; sections were deparaffinized through alcohol gradients and rehydrated to water. Antigens were retrieved by using Citrate pH 6.0 buffer in thermostatic bath at 100°C for 5 minutes. Tissue sections were incubated with primary anti-mouse Ly6G (Biolegend) at the dilution of 1 : 50 or anti-mouse MPO (Abcam) at the dilution of 1 : 200. Rabbit polyclonal IgG (DAKO) was used as negative control instead of primary antibody. Digital imaging was examined using the software LAS V4.5 (Leica DM 2000).

The lesions were removed and cut into pieces. Cell suspensions of skin were prepared after digestion with collagenase I (1 mg/mL, Invitrogen) for 2 h at 37 °C, which was achieved by pressing digested tissue with the cell strainers (70 µm). The single cell suspensions of skin and spleen were marked with the targeted antibodies, including 7-aminoactinomycin D (420403, Biolegend), anti-mouse CD45 (157605, Biolegend), anti-mouse CD3 (100203, Biolegend), anti-mouse CD11b (A15390, eBioscience, CA), anti-mouse F4/80 (25480182, eBioscience), anti-mouse Ly6g (127633, Biolegend) and anti-mouse CD11c (117307, Biolegend). All the samples were harvested by Cytoflex Flow Cytometer (Beckman Coulter, CA).

Antibodies used include: anti-mouse CD45 APCCY7 (Clone 30-F11 Biolegend Cat #103116) and anti-mouse CD45 Eflour 450 (Clone 30-F11 Invitrogen Cat#48-0451-82); anti-mouse LY6G (Clone 1A8; Biolegend Cat#127623); anti-Mouse CD3 Pacific Blue (Clone 145-2C11 Biolegend Cat#100334); anti-Mouse CD3e, PerCP-Cy5.5 (Clone: 145-2C11, eBioscience, Cat#: 45-0031-82); anti-Mouse CD8a PerCP (Clone Ly-2 BDBiosciences Cat#M037858); anti-Mouse CD4, FITC (Clone: GK1.5; eBioscience, Cat#: 25-0041-82); anti-mouse CD19 PE (Clone eBio1D3 ThermoFisher Cat# 12-0193-83); anti-mouse CD11c APC (Clone N418; Biolegen Cat #117310); anti-mouse CD11b FITC (Clone M1/70.15 Invitrogen Cat #RM2801); anti-mouse CD25 PE (Clone 3C7 Biolegend Cat#101904); anti-mouse CD44 PE (Clone 1M7; Biolegend Cat# 10,008); anti-mouse CD62L APC (Clone Mel-14 Biolegend Cat# 104411).

After blocking Fc receptors with anti-mouse CD16/CD32 (BD Biosciences), small intestinal epithelial cells were stained with anti-mouse Ly-6G (BioLegend), anti-mouse F4/80 (BD Biosciences), anti-mouse CD86 (BD Biosciences), anti-mouse CD206 (BD Biosciences), and anti-mouse CD45 (BD Biosciences). The stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using FlowJo Software (FlowJo, Ashland, OR). Neutrophils were defined as Ly6G⁺ cells and macrophages as F4/80⁺ cells and M1 macrophages as F4/80⁺ CD86⁺ and M2 macrophages as F4/80⁺ CD206⁺. Lymphocytes were defined as CD45⁺ cells.