Lsrfortessa x 20 flow cytometer

Flow cytometry detection of C1q and C3b/iC3b/C3dg binding was performed as previously described^{62 (link)}. Tumor cells were suspended in culture media and seeded in 96-well plates at 4 × 10⁴ cells/well at 37 °C, 5% CO₂ for 24 h. Culture media was removed, and cells were treated with Abs in serum-free media at 37 °C, 5% CO₂ for 15 min. NHS (Cedarlane) was added at a final concentration of 25% and  plates were incubated at 37 °C, 5% CO₂ for 15 min. In Supplementary Fig 8a, tumor cells were first incubated with 25% NHS (15 min, 37°C, 5% CO2), followed bytitrated amounts of anti-HER2 or negative control Abs (15 min 37°C, 5% CO2). Following Ab and serum incubation, cells were washed, detached, and resuspended in PBS + 2% FBS and incubated with FITC-conjugated anti-C1q, FITC-conjugated anti-C3/C3b/iC3b/C3dg Abs (clone IH8, specificity C3, C3b, iC3b, C3dg)^{63 (link)} or FITC-conjugated anti-C3/C3b/iC3b (clone 5G9, specificity C3, C3b, iC3b)^{63 (link)} Abs at 4 °C for 1 h. Cells were washed and resuspended in PBS + 2% FBS. The geometric MFI values for the anti-C1q, anti-C3/C3b/iC3b/C3dg or anti-C3/C3b/iC3b Abs were determined using a BD LSRFortessa X-20 Flow Cytometer using FACSDiva v9 and data analysis performed using FlowJo v10. Data was analyzed by non-linear regression using the four-parameter variable slope "log(agonist) vs. response” model in GraphPad Prism 9.2.0.