Fluorochrome conjugated antibodies against cd45

For quantification of sIgA-bound bacteria, a dilution of 10 mg of stool containing approximately 10⁹ cells/mL of Bacteroides were stained on ice with a 1/50 dilution of anti-mouse IgA (eBioscience) in 2% bovine serum albumin (BSA) in PBS. Bacterial cultures were then washed in PBS and analyzed on a CytoFLEX (Beckman-Coulter) using software packages from CellQuest and FlowJo version 11. For host immune cell profiling, approximately 100,000 cells per mouse were stained with fluorochrome-conjugated antibodies against CD45, CD3ε, CD4, CD8, CD38, CD11 b, CD11 c, F4/80, and γδ TCR (eBioscience), and their populations were analyzed by an LSR II flow cytometer (BD Biosciences) and a CytoFLEX (Beckman-Coulter) using software packages from CellQuest and FlowJo version 11.

The suprarenal aorta region from euthanized mice were completely perfused with PBS containing 2% of heparin. Tissues were digested using a cocktail of 450 U/mL collagenase type I (#17100–017, Gibco), 125 U/mL collagenase type XI (#C7657, Sigma-Aldrich), 60 U/mL hyaluronidase type I-s (#H3606, Sigma-Aldrich,) and 60 U/mL DNase-I (#10104159001, Roche) at 37°C for 90 minutes. Single cells filtered through a 70 µm cell strainer were blocked by Fc block (#14–0161-85, eBioscience) for 5 minutes on ice. After blocking, cells were incubated with fluorochrome-conjugated antibodies against CD45 (#48–0451, eBioscience), CD64 (#558455, BD), and CD11c (#47–0114, eBioscience) for 30 minutes on ice, washed, fixed with 2% PFA, and subjected to FACS analysis by the Flow Cytometry Core at the University of Michigan. Data were analyzed using FlowJo v10.6.1.