Complement Deposition Assay for Bacterial Quantification

The complement deposition assay was based on a previously published method^{41 (link)}. Briefly, bacteria was added to brain heart infusion (BHI) broth, incubated at 37 °C for 15 min, and centrifuged. The supernatant was removed and the pellet was washed and re-suspended for incubation in PBS with 20% human serum (pooled from five individuals) and 1% gelatin veronal buffer. After washing, the pellets were re-suspended in mouse-anti-human-C3 in PBS (Abcam) and incubated at 37 °C for 30 min. Washing was repeated, and the contents were re-suspended in anti-Mouse IgG2a-APC in PBS (EBioscience) and incubated at 4 °C for 30 min in the absence of light. After washing, the remaining bacteria were re-suspended in PBS and incubated with thiazole orange (BD Cell Viability kit). Samples were acquired using the Accuri C6 flow cytometer (BD).