SARS-CoV-2 spike proteins induce C5b-9 deposition on the cell surface mainly through the alternative pathway. Flow cytometry demonstrated C5b-9 deposition on TF1PIGAnull cells after adding NHS preincubated with SARS-CoV-2 S1, S2, N, and HCoV-OC43 S proteins (2.5 µg/mL to 20 µg/mL) in either all pathway buffer (GVB⁺⁺ pH 7.4) or APC-specific buffer (GVB⁰ 10 mM MgEGTA pH 6.4). (A) SARS-CoV-2 S1, S2, and HCoV-OC43 S proteins elevated C5b-9 deposition in a dose-dependent manner in GVB⁺⁺ buffer, whereas N protein did not increase C5b-9 from the baseline NHS level. (B) Both SARS-CoV-2 S1 and S2 led to marked increase of C5b-9 depositions in APC-specific buffer. By contrast, SARS-CoV-2 N and HCoV-OC43 S proteins showed minimal C5b-9 increase in APC-specific buffer. All experiments were repeated 6 times. Statistical significance was calculated between each 20 µg/mL CoV protein-treated group and the NHS-treated group (*P < .05, **P < .01, ***P < .001, ****P < .0001).