Anti cd19 bv510 clone 6d5

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).

Splenocytes from immunized mice were plated in a 96-well plate at 10⁶/well in the presence of DMSO or 10 μg/mL GUCY2C _254-262 peptide. Cells were incubated at 37 °C for 1 h, a protein transport inhibitor cocktail (eBioscience) was added, and splenocytes were incubated for an additional 5 h at 37 °C. Cells were then stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen), anti-CD8-PerCP-Cy5.5 (clone 53-6.7; BD Biosciences # 551162; 1:200 dilution), and anti-CD19-BV510 (clone 6D5; Biolegend # 115545; 1:200 dilution). A BD Cytofix/Cytoperm Kit (BD Biosciences) was used for permeabilization and intracellular cytokine staining using anti-IFNγ-PE-CF594 (clone XMG1.2; BD Biosciences # 562333; 1:200 dilution), anti-TNFα-PE-Cy7 (clone MP6-XT22, BD Biosciences # 561041; 1:200 dilution), and anti-MIP1α-APC (clone 39624; R&D Systems # IC450A; 1:200 dilution). Cells were fixed in 2% paraformaldehyde and analyzed on a BD LSR II flow cytometer. Analyses were performed using FlowJo software (TreeStar).