Syto 9 green fluorescent nucleic stain

Feces were collected in Fast Prep lysing Matrix A tubes (MP Biomedicals), resuspended in 1 ml of PBS per 100 mg fecal material, and incubated at 4°C for 20 min. Colon mucosal samples were isolated by removing feces and loose material associated with the colon tissue and scraping along the length of a colon with a cell scraper (Greiner Bio One) into fresh cold PBS. Bacterial suspensions were resuspended in a final volume of 2 ml PBS and incubated at 4°C for 20 min. Samples were homogenized in a FastPrep-24 Tissue homogenizer (MP Biomedicals) for 30 s. After homogenization, samples were centrifuged at 50 × g for 15 min at 4°C to remove debris and the bacteria-containing supernatant transferred through 70 µm filters into a new tube. Bacteria were washed in FACS buffer (PBS, 2% FCS, and 5 mM EDTA) and pelleted at 8,000 × g for 5 min. For flow cytometry, bacterial pellets were resuspended in 100 µl FACs buffer containing SYTO 9 green fluorescent nucleic stain (10 µM; Life Technologies), incubated at 4°C for 15 min, and subsequently stained with 1 µg/ml of an anti-mouse IgA-PE antibody (eBioscience) for 30 min at 4°C. Samples were thoroughly washed before acquisition on the flow cytometer (BD Fortessa).

Feces were collected in Fast Prep lysing Matrix A tubes (MP Biomedicals), resuspended in 1ml of PBS per 100mg fecal material and incubated at 4°C for 20 min. Bacterial suspensions were resuspended in a final volume of 2 ml PBS and incubated at 4°C for 20 min. Samples were homogenized in a FastPrep-24 Tissue homogenizer (MP Biomedicals) for 30s. After homogenization, samples were centrifuged at 50 x g for 15 minutes at 4°C to remove debris and the bacteria-containing supernatant transferred through 70μm filters into a new tube. Bacteria were washed in FACs buffer (PBS, 2% FCS, 5mM EDTA) and pelleted at 8000 x g for 5 min. For flow cytometry, bacterial pellets were resuspended in 100μl FACs buffer containing SYTO 9 green fluorescent nucleic stain (Life Technologies) (10μM), incubated at 4°C for 15 minutes, and subsequently stained with 1μg/ml of an anti-mouse IgA-PE antibody (clone mA-6E1, eBioscience) for 30 min at 4°C. Samples were thoroughly washed and acquired on a BD Fortessa flow cytometer.