Pe conjugated anti human iga

Fecal homogenates were stained with PE-conjugated Anti-Mouse IgA (eBioscience clone mA-6E1) or PE-conjugated Anti-Human IgA (Miltenyi Biotec clone IS11-8E10) prior to flow cytometric analysis or MACS and FACS sorting. See the Extended Experimental Procedures for further information.

Fecal-bound IgA was assessed using bacterial flow cytometry for IgA-bound bacteria (BUGFlow). BUGFlow was performed as previously described (Palm et al., 2014 (link)). In brief, one million isolated fecal bacteria per individual were washed in 1 mL staining buffer (PBS containing 1% (w/v) Bovine Serum Albumin (BSA, Sigma) pelleted (5 min, 8,000x g, 4°C), and blocked for 20 min in 300 μL blocking buffer (20% Normal Mouse Serum (Jackson ImmunoResearch) in staining buffer) on ice. Next, samples were stained with 100ul staining buffer containing PE-conjugated anti-human IgA (1:10, Miltenyi Biotec clone IS11–8E10) or isotype control (mouse IgG1-PE, 1:10, Miltenyi Biotec) for 30 min on ice. Samples were then washed 3 times with staining buffer and fixed in 2% paraformaldehyde (PFA) in PBS at 4°C before flow cytometric analysis on a Beckton Dickinson (Viladomiu et al., 2017 ) LSR Fortessa. Bacteria were gated based on forward and side scatter and the gating strategy was verified by SYTOBC (Invitrogen). An isotype control was used to identify the stained population. Analysis of IgA binding was done using FlowJo (v10.1).