Pe conjugated anti mouse iga

IgA-coated bacteria were quantified as previously described^{28 (link)}. In brief, frozen fecal samples were thoroughly homogenized in PBS to a final concentration of 20 mg/ml. Fecal suspensions were filtered through a 40-μm sterile nylon mesh, then centrifuged at 50×g, for 15 min at 4 °C. 200 μl of supernatant was then washed with 1 ml PBS and centrifuged at 8000×g, for 5 min at 4 °C. Resulting bacterial pellets were resuspended in 100 μl blocking buffer (staining buffer containing 20% Normal Rat Serum) and incubated for 20 min on ice before being stained with 100 μl of staining buffer containing PE-conjugated Anti-Mouse IgA (1:12.5; eBioscience, 12-4204-82) for 30 min on ice, in the dark. Following two washes with staining buffer, pellets were resuspended in 200 μl of FACS buffer (PBS, 1% Normal Rat Serum). Data acquisition was performed on a Beckman Coulter Gallios flow cytometer. For each sample, 50,000 events were recorded and data was analyzed using FlowJo software v.10.8.2. Bacterial load was quantified by gating bacterial population based on size and granularity.

IgA-coated bacteria were isolated and sequenced as previously described^{19 (link),20 (link)}. In brief, frozen fecal samples were thoroughly homogenized in PBS to a final concentration of 100 mg/mL. Fecal suspensions were filtered through a 40-μm sterile nylon mesh, then centrifuged at 50 × g, for 15 min at 4 °C. 100 μL of supernatant was then washed twice with 1 mL of staining buffer (PBS containing 1% (w/v) BSA) and centrifuged at 50 × g, for 15 min at 4 °C. Resulting bacterial pellets were resuspended in 100 μl blocking buffer (staining buffer containing 20% Normal Rat Serum) and incubated for 20 min on ice before being stained with 100 μl of staining buffer containing PE-conjugated Anti-Mouse IgA (1:12.5; eBioscience, 12–4204–82) for 30 min on ice. Following three washes with staining buffer, pellets were resuspended in 200 μL of 0.9%NaCl/0.1 m HEPES buffer (pH 7.2) containing a 1:4000 dilution of SytoBC (Invitrogen, S34855). Data acquisition was performed on a Sony Cell Sorter SH800Z. Samples were gated on appropriate SSC-A/FSC-A gates prior to being selected for SytoBC⁺ events. For each sample, 100,000 events were collected from the IgA⁻ and IgA⁺ population into sterile tubes. Each fraction was stored at − 20 °C prior to DNA extraction and sequencing of bacterial 16 S rRNA genes, as described above.