Fig. 1

Benchmarking a protocol to isolate IgA⁺ and IgA⁻ faecal bacteria by fluorescence activated cell sorting. a IgA-Seq gating strategy. Faecal bacteria from SPF C57BL/6 mice stained with anti-mouse IgA (PE). Gating on a population based on FSC and SSC characteristics, followed by discrimination of SYBR⁺ (FITC) bacteria from autofluorescent (PerCP Cy5.5) debris. IgA⁺ and IgA⁻ populations were sorted from SYBR⁺ cells. b Viability of B. theta VPI-5482 (Bt) collected by various nozzle sizes (μm) and sheath pressures (psi) on the FACSAria III. Culture-grown Bt was stained with mouse anti-Bt IgA (clone 255.4) and rat anti-mouse IgA (PE, clone mA-6E1). One hundred cells were plated in single cell precision mode onto BHIS agar plates for enumeration of colony forming units (CFU). c IgA⁺ and IgA⁻ fractions were collected from SPF C57BL/6J mice (n = 3). Left: percentage of IgA⁺ faecal bacteria detected in each collected fraction for a range of nozzle and pressure configurations. Right: sort purities for these configurations defined by the percentage of events that remained in the designated sort gates. Lines represent means with SEM. dA portion of the IgA-stained faecal suspensions from Fig. 2a were also separated into IgA⁺ and IgA⁻ fractions by magnetic activated cell separation (MACS) as a comparison. PE IgA-stained samples were incubated with anti-PE microbeads and fractions collected by LS column separation. Representative flow cytometry plots of the starting material in SPF stool and the IgA⁺ and IgA⁻ fraction isolated by FACS and MACS. e MACS purity of collected fractions as determined by flow cytometry, technical triplicates from the same stool suspension