Clariostar plus reader

Adapted from Tang et al.³⁸ , primary macrophages were treated as indicated in 12-well plates in DMEM containing 20% L929 conditioned medium. Following treatments, supernatant was removed and 200 µL of cell culture grade water was added to each well and plates were incubated for 15 m at 37 °C for lysis. The lysates were then transferred to 1.5 mL Eppendorf tubes and centrifuged at 10,000 × g for 5 m. 50 µL of lysate was then mixed with 50 µL MQAE (10 µM) and added to a black 96-well plate. The fluorescence intensity was measured at Excitation 350 nm and Emission 460 nm using the BMG Labtech CLARIOstar^Plus reader.

Cell metabolism was studied by subjecting the cells to a colorimetric assay that uses Alamar Blue reagent. This assay is used to quantify the number of cells with active metabolism and is based on the reduction of resazurin, a non-fluorescent indicator which is metabolised to its fluorescent metabolite resorufin by viable cells in culture. At the end of the exposure time (i.e. 3, 6, 12, 24 or 48 hours), 5 μL of Alamar Blue (Thermo Fisher Scientific) was added to each well and incubated for 1 hour. The fluorescence was subsequently measured using a CLARIOstar Plus reader (BMG Labtech) at 540 nm for excitation and 590 nm for emission. Cell metabolism was calculated by comparing the fluorescent values of the venom samples with those of the negative control (i.e. average signal without venom, where normal metabolic activity is expected).

Serial dilutions of serum samples were prepared, mixed with S⁺ VLPs, preincubated for 30 min, and incubated with U251MG (hACE2⁺ and NM∼LgBiT⁺) recipient cells at 37°C for 4 h. Upon replacement of the supernatant with substrate, bioluminescence was immediately quantified in a CLARIOstar Plus reader (BMG Labtech). SI Materials and Methods and Figure S6 (Supplementary Material) provide a detailed protocol.

Cell viability was quantified using ATP-levels in cell lysates. As ATP is involved in a variety of enzymatic reactions and ATP-levels decline rapidly when cells undergo necrosis or apoptosis, this assay is widely accepted as a valid marker of viable cells, as higher ATP-concentrations indicates a higher number of living cells. This bioluminescent assay uses ATP from viable cells to generate photons of light. ATP-levels were measured using the ATPlite kit from PerkinElmer (6016731) according to the manufacturer’s instructions. After the cells were incubated with the venom for 24 or 48 hours, cells were subjected to this assay after the luminescence was measured on a CLARIOstar Plus reader (BMG Labtech). Cell viability was subsequently quantified by comparing the ATP-levels in venom-incubated wells with the ATP-levels of the negative control (where cells were 100% viable).

The thromboxane synthase (TxS) activity was evaluated as previously described [109 (link),110 (link)]. Briefly, WP (6 × 10⁸ cells/mL) were incubated with gossypol (40 μM; 10 min) or the flavonoid aglycones (100 μM), in the presence or absence of nonselective cyclooxygenase (COX-1 and COX-2) inhibitor indomethacin (10 μM) at 37 °C for 30 min. DMSO was added to control samples as a vehicle of the flavonoid aglycones. Platelets were stimulated with thrombin (50 mU/mL), and the reaction was stopped by trichloroacetic acid (TCA; 20% TCA in 0.6 M HCl). Then, samples were incubated on ice (10 min) and centrifuged (4 °C, 10 min, 4.400 g). The supernatant was mixed with thiobarbituric acid (TBA; 0.53% TBA in 0.01 M KH₂PO₄, 0.05 M Na₂HPO₄, pH 7.4), heated at 70 °C for 30 min, and cooled at RT. Then, the fluorescence of the reaction product of malondialdehyde (MDA) and TBA was measured (λex = 510 ± 15 nm, λem = 560 ± 20 nm, CLARIOstarPlus reader, BMG Labtech Gmbh, Ortenberg, Germany). To evaluate the TxS activity, the exhibited fluorescence of the samples in the presence of indomethacin was indicated with ethanol (the vehicle for indomethacin).