Profiling Gut Immunoglobulin-Coated Bacteria

Fecal flow cytometry and IgA-Seq were performed essentially as previously described except that Ig-coated bacteria were isolated using MACS beads and a custom-built 96 well magnetic separator (https://www.kjmagnetics.com/proddetail.asp?prod=D28-N52) followed by negative selection using MACS multi 96 columns^{21 (link)}. Bacteria were stained with either PE-conjugated Anti-Human IgA (1:10; Miltenyi Biotec clone IS11-8E10) or FITC- or PE- conjugated Anti-Human IgM (1:50 JI 109-096-008). IgM-Seq samples were processed identically to IgA-Seq except that PE-anti-IgM was used in place of PE-anti-IgA. The estimated bacterial numbers in all samples post-separation exceeded 1 × 10⁷ bacteria per fraction^{21 (link)}.