Apc eflour780 anti mouse cd4 gk1

LECs in phosphate-buffered saline (PBS) containing 0.2 mM calcium, 0.1 mM magnesium, and 0.5% w/v bovine serum albumin (BSA) were treated with anti-CD16/32 (clone 93; eBioscience) to block Fc receptors, and then stained with antibodies to cell surface molecules in the same buffer. Flow cytometry antibody used was: APC-eflour780-anti-mouse CD4 (GK1.5; eBioscience); PE-anti-mouse CD25 (PC61.5; eBioscience); PE-anti-mouse CD44 (IM7; eBioscience); APC-anti-mouse VCAM-1 (clone 647; eBioscience). For detection of LTα2β1, T cells were incubated with MOPC21 or LTβRIg at 2 μg /mL for 60 min at 37 °C in HBSS with 2% FBS. Cells were washed, and then stained for 45 min at 4 °C with BV421-rat anti-mouse IgG1 along with antibodies to other cell surface markers. Cells were then washed and fixed with 4% paraformaldehyde and run on an LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA). Results were analyzed with FlowJo 8.7 (Treestar). Flow cytometry gating information is included in Supplementary Fig 8.