Microbiome frozen in 50% glycerol were defrosted on ice before being
washed in PBS and pelleted for 3 minutes at 8,000 rpm. Bacteria were then
stained on ice for 30 minutes with IgG-PE (Biolegend, clone HP6017, cat. 409304;
dilution 1:100), IgA1/2 biotin (BD, clone G20-359, cat. 555884; dilution 1:50),
followed by 20 minutes with streptavidin-APC (Biolegend, cat. 405207; dilution
1:100). For isotype controls, mouse IgG1 κ Isotype- Biotin (BD, cat.
550615; dilution 1:50) or mouse IgG2a, κ Isotype- PE (Biolegend, cat.
400212; dilution 1:100) were used. The bacteria were washed before and after DNA
was stained with Hoechst33342 (Sigma-Aldrich). The stained bacteria were sorted
as Hoechst+ and IgG+IgA+ or
IgG−IgA+ into PBS using the BD Influx and then
stored at -80°C until DNA extraction.
washed in PBS and pelleted for 3 minutes at 8,000 rpm. Bacteria were then
stained on ice for 30 minutes with IgG-PE (Biolegend, clone HP6017, cat. 409304;
dilution 1:100), IgA1/2 biotin (BD, clone G20-359, cat. 555884; dilution 1:50),
followed by 20 minutes with streptavidin-APC (Biolegend, cat. 405207; dilution
1:100). For isotype controls, mouse IgG1 κ Isotype- Biotin (BD, cat.
550615; dilution 1:50) or mouse IgG2a, κ Isotype- PE (Biolegend, cat.
400212; dilution 1:100) were used. The bacteria were washed before and after DNA
was stained with Hoechst33342 (Sigma-Aldrich). The stained bacteria were sorted
as Hoechst+ and IgG+IgA+ or
IgG−IgA+ into PBS using the BD Influx and then
stored at -80°C until DNA extraction.