Endogro mv culture medium

Bacteria were grown to an OD₆₀₀ of 0.25 in EndoGRO-MV culture medium (Millipore), the bacteria suspension was smeared on charged glass slides (Fisher), and the slides were fixed with 4% paraformaldehyde for 30 min at room temperature. The slides were blocked with 3% BSA for 1 hour, then incubated with rabbit antibodies to BspC or IgG at a 1:50 dilution followed by donkey anti-rabbit conjugated to AlexaFluor488 (Invitrogen). Bacteria were imaged using a BZ-X710 fluorescent microscope (Keyence).

The anti-BspC polyclonal antibody and an IgG antibody control (Invitrogen) were diluted to 2.28 mg/mL and adsorbed (as previously described [6 (link)]) against COH1ΔbspC bacteria to remove natural rabbit antibodies that react non-specifically with bacterial surface antigens by incubating with COH1ΔbspC overnight at 4°C, with rotation. Bacteria were pelleted by centrifugation and the supernatant was collected and filtered using 0.22 μM cellulose acetate SpinX centrifuge tube filters (Costar).
In order to assess surface expression of WT or mutant BspC, GBS strains were grown to OD600 of 0.25 in EndoGRO-MV culture medium (Millipore) to mimic host infection conditions, pelleted by centrifugation, and resuspended in PBS. Approximately, 1 x 10⁶ CFU of each strain was incubated with either adsorbed anti-BspC antibody or adsorbed anti-rabbit IgG at a 1:50 dilution at 4°C, overnight, with rotation. The next day, bacteria were washed via centrifugation, and labeled with a donkey anti-rabbit IgG conjugated to AlexaFluor488 (Invitrogen) at a 1:2,000 dilution for 45 minutes at room temperature with rotation. Samples were washed again and then resuspended and read on a FACScalibur flow cytometer (BD Biosciences) and analyzed using FlowJo (v10) software.