α mouse iga pe

Pre-weighed frozen fecal samples in glycerol were thawed at 4°C and then homogenized using a handheld homogenizer and pestles (Kimble Chase Kontes) at a final dilution in sterile PBS of 100 mg per ml. Samples were processed as described previously (Palm et al., 2014 (link)). After homogenization, samples were centrifuged at 50 × g at 4°C for 15 minutes, then washed three times in 1 ml PBS/1% BSA at 8000 × g for 5 minutes each. The pre-sort fraction was collected as 20 μL after resuspension prior to the final wash and stored at −80°C. The cell pellet was resuspended in 25 μL of 20% Normal Rat Serum (Jackson Immunoresearch) in PBS/1%BSA and incubated for 20 minutes on ice. After incubation, 25 μL 1:12.5 α-mouse- IgA-PE (EBioscience, clone mA-6E1) was added to each sample and samples incubated on ice for 30 minutes. Samples were washed three times in 1 ml PBS/1% BSA as above, and finally resuspended in PBS/1% BSA and transferred to blue filter cap tubes (VWR 21008–948) for flow sorting. An average of 500,000 cells from the IgA-positive and IgA-negative bacteria were sorted in triplicate into sterile microcentrifuge tubes on the BD FACSAria II at the MIT Koch Institute Flow Cytometry Core (Cambridge, MA). Samples were centrifuged, supernatant removed, and stored at −80°C until nucleic acid extraction using the DNeasy UltraClean Microbial Kit (Qiagen).