Anti siglec f pe cf594 clone e50 2440

Peritoneal cells from Scn8a^dmu/+ and Scn8a^+/+ mice (n = 16 each) were harvested by i.p. injection and recovery of 4 ml PBS, 0.5% bovine serum albumin, 5 mM EDTA. Mouse peritoneal cavity cells (PCC) were counted. Cells were centrifuged at 400 x g for 5 minutes at 4°C and resuspended in a 1/1000 dilution of fixable viability dye (FVD) eFluor 450 (eBiosciences) in PBS for 20 minutes. Then the cells were washed with wash buffer (PBS + 2% FBS + 20 mM NaN₃), centrifuged and resuspended in antibody cocktails containing anti-CD19-BV510 (clone 6D5, BioLegend), anti-CD11b-PE-eFL610 (clone M1/70, eBiosciences), CD117 (c‐kit)‐PE (clone 2B8, BioLegend), anti-CD4-APC (clone GK1.5, eBiosciences), anti-FcϵRI-APC eFL780 (clone Mar-1, eBioscience), anti-CD8a-BV650 (clone 53-6.7, BD Biosciences), anti-CD3-BV711 (clone 145-2C11, BD Biosciences), anti-Siglec-F-PE-CF594 (clone E50-2440, BD Bioscience), anti-CD11b-PE (clone M1/70, eBiosciences), anti-F4/80-PE-Cy7 (clone BM8, eBioscience), anti-Ly6C-APC-eF780 (clone HK1.4, eBioscience), or anti-Ly6G-BV605 (clone 1A8, BD Biosciences) for 30 minutes. Stained fixed cells were acquired for analysis using a BD LSRFortessa and results were analyzed using FlowJo (Ashland, OR) software. A visual representation of the gating strategy used to identify the cell populations in the mouse peritoneum is provided (Supplementary Figure 2).

50 μL of whole blood collected in heparin tubes (BD) was incubated with fluorochrome tagged antibodies at 4°C for 30 minutes. Antibodies for the innate panel: anti-CD11b–Pacific Blue (clone M1/70, eBioscience), anti-CD11c–PE-Cy7 (clone HL3, BD Bioscience), anti-Siglec-F–PE-CF594 (clone E50–2440, BD Biosciences), anti-F4/80–APC (clone BM8, eBioscience), anti-Ly6C–FITC (clone AL-21, BD Bioscience), anti-Ly6G–PerCP–eFluor710 (clone 1A8, eBioscience). Antibodies for the adaptive panel: anti-CD3–PE (clone145-2C11, eBioscience), anti-CD19–PE-Cy7 (clone eBio1D3 (1D3), eBioscience), anti-CD4–FITC (clone GK1.5, eBioscience), anti-CD8 AF700 (clone 53-6.7, BD Bioscience). After RBC lysis, cells were subsequently washed with PBS/0.5% BSA and resuspended in 1% paraformaldehyde.
If required, panels were modified to contain anti-CD45.1–APC (clone A20, BD Bioscience, 1:100) and anti-CD45.2–BUV395 (clone 104, BD Bioscience, 1:100).
Cells were acquired on the Fortessa-X20 (BD) and analyzed using FlowJo software (version 10.6.1). All percentages are of single viable frequency, unless otherwise indicated.