Nunc 96 well polystyrene round bottom microwell plates

The SBA assay was performed to evaluate the complement-mediated killing features of the anti-PspA_1-5c+p antibody against three strains of the pneumococcus expressing two families of the PspA. For this purpose, Thermo Scientific Nunc™ 96-Well Polystyrene Round Bottom microwell plates were coated with 12.5 μl of the three strains of pneumococcus at 10⁵ CFU/ml (based on the standard of 0.5 McFarland) separately, and 12.5 μl of diluted inactivated serum sample at 56 °C for 30 min (1:2 to 1:64). Afterward, fresh infant rabbit serum (4%) was added to each well as a source of the complement. At two intervals (0 and 2 h), the sample from each well was cultured in blood agar media. After 18–24 h incubation at 37 °C in 5% CO₂, the colony-forming unit of the bacteria was counted. The wells containing bacteria and rabbit complement were used as a negative control [64 (link), 65 (link)].

Ex vivo cultures were prepared as described previously^{38 (link)}. Briefly, patient tumor tissues were mechanically disaggregated and treated for 1 h with 125 U/mL collagenase and 2.5 mg/mL DNAse (both from Sigma-Aldrich). To remove aggregates and debris the cell suspensions were filtered through 100 μM nylon Cell Strainer (BD Falcon, Franklin Lakes, NJ) and washed with ice-cold PBS. Red blood cells were removed using ACK lysing buffer (Lonza, Basel, Switzerland). Live cells were seeded out at density of 15–20 × 10⁵ cells/well in Nunc™ 96-Well Polystyrene Round Bottom Microwell plates (Thermo Fisher Scientific, Waltham, MA) in RPMI++ medium supplemented with 100 units/mL penicillin and 0.1 mg/mL streptomycin (both from Lonza) and allowed to form 3D spheroids. Drugs were immediately added to the cells and left for five days before viability was assessed using the CTG Luminescent Cell Viability Assay (Promega) and analyzed by Fluoroscan Ascent Fl (Thermo Fisher Scientific). The ex-vivo assay was performed once for each patient sample, with at least three technical replicates per condition.