Non enzymatic cell stripper liquid

For in vitro experiments, BMDMs and BMDCs differentiated for 7 days were seeded at 1×10 6 cells/well in a 12-well dish. Cells were treated with LPS, and 0 and 24 hours and after incubation cells were removed using non-enzymatic cell stripper liquid (Corning Inc., Corning, NY) and subjected to staining for flow cytometry. For in vivo experiments, spleen and lymph nodes were isolated from mice single cell suspensions were prepared followed by passing through a 70 μm strainer. Red blood cell (RBC) lysis was performed by incubating the cells in 0.8% ammonium chloride solution (StemCell Technologies, Cambridge, MA) and cells were subjected to staining for flow cytometry analysis. Cells were stained with the UV LIVE/DEAD fixable stain (Invitrogen) and then surface labeled for different combinations of following markers: CD11b, CD11c, CD80, CD86, CD40, CD19, TCRβ, Ly6G, MHCII, CD4, and CD8 (Biolegend, San Diego, CA) and fixed with 1% paraformaldehyde (Sigma Aldrich, St. Louis, MO). Samples were analyzed on an LSRII cytometer (BD Biosciences) or an Aurora (Cytek Biosciences). All flow cytometry data analysis was performed using FlowJo Software version 10.6.1 (BD Biosciences).