Cytotox glo cytotoxicity assay reagent

HEK293-TLR8 cells were seeded on a 96-well plate at a cell density of 1 × 10⁴ cells/mL and was grown for 24 h in DMEM with 10 % FBS. Cells were then co cultured with all available OMVs (Additional file 1) complexes per well (50 μl OMV [OD₆₀₀ 0.8]/well). Fifty microliter of CytoTox-Glo™ Cytotoxicity Assay Reagent (Promega, USA) per well (12.5 μl for a 384-well plate) of HEK293-TLR8 was also added and luminescence was measured after incubating for 15 minutes at room temperature. After that, 50 μl of lysis reagent was added to all wells (12.5 μl for a 384-well plate), mixed and incubated at room temperature for 15 minutes. Then again, luminescence was measured and the viable cell luminescence was calculated as manufacturer’s instruction.

Ramos cells were washed with GVB++ (Complement Technology, B100) and seeded at 1 × 10⁵ cells per well in a 96-well white clear-bottom polystyrene plate (Costar, 3610), to which 50 µL of 10 µg/mL of rituximab (AbMole Bioscience, M5219), rhesusized rituximab (NHPRR, 2B8R1), or GVB++ was added. The plate was subjected to orbital shaking at 200 rpm for 2 min. Following antibody binding, 50 µL of a 1:100 dilution of serum was added. After repeating the shaking procedure, the plate was incubated at 37°C in a 5% CO₂ incubator for 1 hour and iced for 10 min, and then, 50 µL of CytoTox- Glo Cytotoxicity assay reagent (Promega, G9291) was added. The plate was shaken at 900 rpm for 1 min on an orbital shaker and then incubated for 15 min at room temperature. Luminescence dependent on serum complement was measured using SpectraMax Paradigm Plate reader, and total luminescence was measured following addition of 50 µL of lysis reagent and incubation at room temperature for 15 min. Relative antibody-dependent lytic activity was calculated as the ratio of luminescence of antibody-dependent complement-mediated cell death to luminescence observed for the no-antibody control condition. Mean values of assay duplicates were reported.