Cd24 buv 737

BMDCs and/or whole lung lysates were incubated with Zombie UV Fixable Viability Kit (BioLegend, San Diego, CA) at room temperature for 15 minutes. Cells were washed with FACS rinsing buffer, pelleted, and incubated with anti-mouse CD16/32 (BioLegend) for 15 minutes at 4 °C to block non-specific Ig binding. Following subsequent washing and centrifugation, cells were incubated with antibody cocktails containing some or all of the following markers: CD11c BV711, CD80 BV650, CD64 PerCP/Cy5.5, Ly6C BV605, CCR-7 APC, MHC-II (I-A/I-E) BV 421, Ly6G BV650, CD40 PE/Cy7,CD4 APC/Cy7, CD3 APC, and CD8α PerCP/Cy5.5 (BioLegend); CD86 BUV395, CD11b BV480, Siglec-F APC-R700, CD45 BV805, CD103 PE-CF594, and CD24 BUV 737 (BD Biosciences, San Jose, CA). Cells were analyzed using the BD LSRFortessa™ (BD Biosciences). Cell aggregates were removed by gating single cells on the forward light scatter (FSC-H vs FSC-A). Dead cell and debris were excluded before gating for myeloid innate immune cells and/or T-cell markers. Fluorescence minus one (FMO) controls were used to set up gating strategies used in these experiments. Data was analyzed using FlowJo software v10.7.1 (Tree Star Inc.).