Synergy ht multi detection

At 24, 48, 72, 96, and 120 h PI, supernatant of all conditions was collected and stored at -20°C for LDH (Lactate Dehydrogenase) quantification using the LDH Liquiform kit according to manufacturer instructions. Initial absorbance was registered after 1 min and the second was registered after 2 min from the first one. Absorbances were conducted in BioTek Synergy HT Multi-Detection (Winooski, VT) with 340 nm. Results were expressed in U/L.

For this assay, the Pierce™ Chromogenic Endotoxin Quant Kit was used. The plate was pre-warmed to 37 °C, followed by the addition of 50 µL of CVX and standards. Then, 50 µL of lysate was added, and incubated at 37 °C for 30 min. After 30 min, 100 µL of chromogenic substrate was added, and incubated at 37 °C for 6 min. After the reaction, the stopping solution was added. Absorbance was measured spectrophotometrically (Bio-Tek Synergy HT Multi-Detection, Winooski, VT) at 405 nm. Data were expressed in EU/mL. 0.5 EU/mL of endotoxin was detected in the CVX preparation. The sample used is within the acceptable threshold of 1 EU/mL according to Pinto et al.⁵² .

TG-macrophages (2x10⁵) obtained according to item 2.6 were incubated with RPMI (negative control), BjV (25 μg/mL), and BthTX-I or BthTX-II (25 μg/mL) for 90 min, at 37°C in a humid atmosphere (5% CO₂) in the presence or absence of LED photobiomodulation. After this period, the cells were centrifuged for 1100 xg for 15 min at 4°C and the SOD, catalase, and peroxidase enzymatic activities were determined by commercial kits from Abcam (Cambridge, UK). Absorbances and fluorescences were determined in BioTek Synergy HT MultiDetection (Winooski, VT) at 450 nm and excitation/emission 535/587 nm and expressed in μm/mL or Pm/mL [21 (link), 22 (link)].

The cytotoxicity of lupane against PBMCs was determined by an MTT assay [19 (link)], which analyzes the ability of living cells to reduce the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product. Cells were plated in 96-well plates (2 × 10⁵/well) and maintained in RPMI assay medium (RPMI-1640 medium supplemented with 100 μg/mL of gentamicin, 2 mM of L-glutamine and 10 % FBS). PBMCs were incubated with RPMI (negative control), RPMI plus 2 % ethanol (diluent control) or different concentrations of lupane (0.3–12 μg/mL), diluted in ethanol, at 37 °C, under an atmosphere of 5 % CO₂ for 1, 15 and 24 h. After incubation, the supernatant was replaced by a fresh medium containing MTT (0.5 μg/mL). Four hours later, the plates were washed 3 times with PBS, the formazan crystals were dissolved in 100 μL DMSO and the absorbance was measured with the Bio-Tek Synergy HT Multi-Detection (Winooski, VT) at 540 nm.

A fluorescent substrate 2′ (4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA; Sigma) was used to measure NA enzymatic activity, according to the manufacturer’s recommendations. Viruses were prepared using serial twofold dilutions in 50 μl of calcium saline buffer in 96-well plates and 50 μl of 200 μmol MUN was added to each well, and then incubated for 60 min at 37°C in darkness. One hour later, stop solution was added and NA activity was quantified using a Synergy HT Multi-Detection microplate reader (BioTek, Winooski, VT, United States) with excitation and emission wavelength of 360 and 440 nm.

In order to determine the effective non-toxic extract concentrations, the cytotoxicity of the extracts was monitored through the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, following a methodology previously described [25 (link)]. The assay consisted of the reduction of the yellow MTT to insoluble purple formazan crystals by dehydrogenizing metabolically active cells. After the incubation period of 24 h, 100 µL of MTT solution (0.5 mg/mL), freshly prepared in DMEM at 37 °C, was added to each well and incubated at 37 °C for 45 min. After the incubation period, the supernatant was removed and the resulting formazan crystals were dissolved in 100 µL DMSO. The absorbance of the colored product was determined at 515 nm using a Synergy HT Multi-Detection microplate reader (Biotek, Germany) operated by GEN5 software. Cytotoxicity was expressed as the percentage of cell viability vs. the control (0.25% DMSO). At least four independent assays were carried out in duplicate.