Brilliant violet 421 anti rabbit igg

1 μl of overnight cultures made in 0.2μm-filtered LB, or 1μl of fresh feces or cecal content suspension (as above) was stained with 0.2μm-filtered solutions of STA5 (human recombinant monoclonal IgG2 anti-O:12-0, 6μg/ml ^{15 (link)}), Rabbit anti-Salmonella O:5 (Difco, 1:200) or Rabbit anti-Salmonella O:4 (Difco, 1:5). After incubation at 4°C for 30 min, bacteria were washed twice by centrifugation at 7000g and resuspension in PBS/1% BSA. Bacteria were then resuspended in 0.2μm-filtered solutions of appropriate secondary reagents (Alexa 647-anti-human IgG, Jackson Immunoresearch 1:200, Brilliant Violet 421-anti-Rabbit IgG, Biolegend 1:200). This was incubated for 10-60 min before cells were washed as above and resuspended for acquisition on a BD LSRII or Beckman Coulter Cytoflex S. A media-only sample was run on identical settings to ensure that the flow cytometer was sufficiently clean to identify bacteria without the need for DNA dyes. Median fluorescence intensity corresponding to O:12-0 or O:5 staining was calculated using FlowJo (Treestar, USA). Gates used to calculate the % of “ON” and “OFF” cells were set by gating on samples with known O:5/O:4 (oafA-deletion) and O:12-0 (gtrC-deletion) versus O:12-2 (pgtrABC) phenotypes (Fig. S2 and 3).