Bacterial flow cytometry (BUGFlow)

Bacterial flow cytometry (BUGFlow)

Bacterial flow cytometry (BUGFlow) was performed as previously described (17). Briefly, 1 million isolated fecal bacteria or bacterial strains were stained in staining buffer [PBS containing 1% (w/v) BSA (Sigma)]. For sorting of IgA-bound bacteria, autologous gut bacteria were blocked for 20  min with subsequent incubation of 30 min with phycoerythrin (PE)–conjugated anti-human IgA (1:10; Miltenyi Biotec, clone IS11-8E10) or the respective isotype control (1:10; Miltenyi Biotec, mouse IgG1). For binding assays with CSF or recombinant antibodies, samples were incubated for 1 hour on ice with CSF supernatant (1:5), patient-derived recombinant antibodies (dilution series), or rabbit anti–Escherichia coli (polyclonal, 1:100; Abcam) (positive control) followed by washing and staining with buffer containing PE-conjugated anti-human IgG (clone HP6017, 1:20; BioLegend), PE-conjugated anti-rabbit IgG (polyclonal, 1:1000; Abcam), or respective isotype control for 30 min on ice. Samples were then washed three times before flow cytometric sorting on FACSAria II (Beckton Dickinson) or fixed with 2% paraformalde-hyde before flow cytometric analysis on LSRFortessa (Beckton Dickinson). Bacteria were gated on the basis of forward and side scatter, and the gating strategy was verified by SYTO BC (Invitrogen). An isotype control was used to identify the stained population. For CSF binding, samples that showed reactivity in two independent experiments were considered positive.

In case of subsequent sorting, we collected 1 million events from the IgA+ population and at least 100,000 commensal microbial cells from the IgA− input into sterile sorting buffer. Each fraction was stored at −80°C before amplification and sequencing of bacterial 16S rRNA genes. Analysis of IgA binding was done using FlowJo software (v10.1). Multiple precautions were taken to minimize potential contamination of sorted fractions as previously reported (19): (i) Freshly autoclaved PBS was used for sheath fluid, (ii) the flow cytometer was sterilized according to the manufacturer’s recommended protocol, (iii) the sheath fluid filter was replaced routinely, and (iv) 16S rRNA analysis was performed on samples collected from the flow cytometer droplet stream before and after every sort as well as from the reagents used (mock sample without bacteria), thus permitting identification of any sequences that did not originate from the sorted sample.