Phenol free rpmi medium

S. aureus Newman Δspa was grown in phenol-free RPMI medium (Gibco) at 37 °C overnight. The bacteria were washed two times with PBS and resuspended in 0.05% bovine serum albumin (BSA) in phenol-free RPMI to an OD₆₀₀ ~ 1. 500 μl of bacteria was mixed with 500 μl of NHS (BioIVT) or 500 μl 0.05% BSA in phenol-free RPMI. The samples were incubated at 37 °C for 8 h with shaking. The bacteria were then washed three times with PBS. For EM analyses, samples were fixed 1:1 with a 2x fixative [5% glutaraldehyde, 4% paraformaldehyde (PFA) in 0.2 M sodium cacodylate buffer] and dehydrated through a graded series of ethanol. Samples were critical point dried using liquid carbon dioxide in a Tousimis Samdri 795 Critical Point Drier, coated with carbon in a Quorum EMS 150T ES (Quorum Technologies Ltd) and examined in a Zeiss Supra Field Emission Scanning Electron Microscope (Carl Zeiss Microscopy), using an accelerating voltage of 8 KV.

To remove IgG from serum, S. aureus was added to a final OD₆₀₀ of 1.0 to NHS and incubated on ice for 30 min. The sample was centrifuged, and the pellet was discarded. Fresh S. aureus was added, and the serum was again incubated on ice for 30 min. After centrifugation, the supernatant was used as “preadsorbed serum.” S. aureus Newman Δspa was grown in phenol-free RPMI medium (Gibco) to an OD₆₀₀ ~1.0, washed 1x with PBS and resuspended in 3% BSA in PBS. Bacteria blocked for 30 min at room temperature (RT) and washed 2x with PBS were resuspended in 0.05% BSA in phenol-free RPMI to an OD₆₀₀ ~ 1. 200 μl of bacteria was mixed with 200 μl of NHS (Quidel), preadsorbed serum, C1q-depleted human serum (Quidel), or media plus antibody at 100 μg/mL final concentrations and incubated for 1 h at 37 °C with shaking. After incubation, samples were washed 2x with 1% BSA in PBS and resuspended in 1% BSA in PBS containing 3 μg/ml anti-C3b antibody (clone 3E7; Biolegend). After incubating for 1 h at RT and washing 2x with 1% BSA in PBS, samples were resuspended in 1% BSA in PBS containing 2 μg/ml goat anti-mouse IgG antibody, Alexa Fluor 488 conjugated (Invitrogen) and incubated at RT for 45 min. 0.5 μM ToPro3 (ThermoFisher) was added for 15 min. Samples washed 2x with 1% BSA in PBS were resuspended in 1% BSA in PBS and analyzed on a BD FACSCanto II.

S. aureus Newman Δspa was grown in phenol-free RPMI medium (Gibco) at 37 °C overnight. The bacteria were washed once with PBS and resuspended in 3% BSA in PBS. They were blocked for 30 min at room temperature, washed twice with PBS and resuspended in 0.05% BSA in phenol-free RPMI to an OD₆₀₀ ~ 1. 500 μl of bacteria was mixed with 500 μl of 20% NHS (BioIVT), 500 μl 20% C6-depleted serum (Quidel) or 500 μl 0.05% BSA in phenol-free RPMI. The samples were incubated for 1 h at 37 °C with shaking, then the bacteria were washed three times with PBS and resuspended in 1 ml of 1% BSA in PBS. Samples were aliquoted and incubated with goat anti-human C1q (Genway Biotech, Inc.), mouse anti-human C3 (Biolegend) or mouse anti-human C5b9 (Abcam) (at a final concentration of 1 μg/ml) for 1 h at room temperature and then washed three times with PBS. There were resuspended in 100 μl of 1% BSA in PBS and then probed with donkey anti-goat IgG conjugated to Alexa Fluor 488 (1:4000; ThermoFisher). Mouse IgG1 and mouse IgG2a antibodies (produced at Regeneron) against non-complement antigens were used as controls. DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride, Biolegend) was added to samples at 5 μg/ml and they were incubated for 45 min at room temperature. The samples were then visualized on a Zeiss LSM780 confocal microscope.