Cd11c pe cy7 hl3

Spleens were perfused with collagenase D (1 mg/ml) and DNAse I (0.1 mg/ml), cut into small pieces and incubated at 37°C for 45 min. Cell preparations were filtered using 100‐μM cell strainers, and APC populations were enriched using magnetic depletion of B, NK, and T cells (Miltenyi). For experiments that focused only on cDC, cDC were enriched using CD11c‐positive selection (Miltenyi). Cell suspensions were labeled with the following antibodies: CD3‐APC (145‐2C11, 1/300 eBioscience), CD19‐APC (ID3, 1/300 Biolegend), NK1.1‐APC (PK136, 1/300 BD Pharmingen), B220‐BV510 (RA3‐6B2, 1/500, BD Horizon), CD11b PE‐CF594 (M1/70, 1/3000 BD Horizon), CD11c PE‐Cy7 (HL3, 1/400 BD Pharmingen), Ly6C‐PerCP‐Cy5.5 (AL‐21, 1/1000 BD Pharmingen), CD64 BV421 (X54‐5/7.1, 1/300 Biolegend), CD8α APC‐Cy7 (53‐6.7, 1/200 BD Pharmingen), or XCR1‐PE (REA707, 1/100 Miltenyi). Cells were sorted on a BD AriaSorp (BD Biosciences) with purity routinely higher than 95%. Expression of MHC II was assessed in a separate mix since the anti‐I‐A^b AF700 (M5/114, 1/500 BD Pharmingen) is a blocking antibody. For experiments with Karma mice in which cDC1 are Tomato⁺, CD11b‐PerCP‐Cy5 (M1/70, 1/200 BD Pharmingen) was used. All flow cytometry samples were run on a Fortessa (BD Biosciences) and analyzed using FlowJo software.

Blood samples (~500μl) were immediately lysed, washed, suspended in PBS and incubated with Aqua vital dye to distinguish live from dead cells (Invitrogen, Burlington, ON, Canada). Following two more washes, cells were suspended in washing and staining buffer (1% bovine serum albumin-PBS) and incubated for 20 minutes with the following cocktail of pre-titrated anti-mouse antibodies: CD3-Vioblue (145-2C11), CD4-APC-H7 (GK1.5), CD62L-PE (MEL-14-H2.100) (Miltenyi Biotec), CD44-Brilliant Violet 570 (IM7; BioLegend, San Diego, CA), CD11c-PE-Cy7 (HL3; BD Biosciences) and CCR5-FITC (CTC5), CXCR6-PerCP (221002) and CCR2-APC (475301) (R&D Systems Inc., Minneapolis, MN). All events were acquired in a BD FACSAria^™ Cell Sorter and analyzed with FlowJo vX.0.7 software (TreeStar, Ashland, OR).