Alexa647 labelled donkey antimouse igg

Bacteria were grown in Tryptic Soy Broth to OD₆₂₀ ~ 0.23, washed once with HBSS + Ca²⁺/Mg²⁺ + 0.1% (w/v) gelatin (HBSS3+) and diluted to an OD₆₂₀ of 0.2 with HBSS3+. Twenty‐five microlitre bacteria was mixed with 25 μL 20% (v/v) patient serum diluted in HBSS3+ with or without reconstitution of 0.2 μg mL⁻¹ FD (CompTech) and/or 30 μg mL⁻¹ anti‐C1q and incubated 30 min at 37°C. Bacteria were pelleted by centrifugation at 3200× g and fixed for 20 min in 2% (w/v) paraformaldehyde in PBS at RT. Surface‐bound complement C3 and complement complex C5b‐9 were detected with 1:500‐diluted FITC‐labelled polyclonal goat anti‐human C3 (MP biomedicals) and 1:100‐diluted monoclonal mouse antihuman C5b‐9 (Clone aE11, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by 1:500‐diluted Alexa647‐labelled donkey antimouse IgG (Invitrogen). Surface binding of C3 and C5b‐9 was determined by flow cytometry using a FACS LSR II instrument (BD Biosciences, San Jose, CA, USA) and expressed in mean fluorescence intensity (MFI) in arbitrary units (AU). Data were analysed by using FlowJo version 10.4.1 (BD Biosciences).