Victor nivo plate reader

All growth curves were performed in 96-well format in an anaerobic chamber (95% N₂, 5% CO₂ atmosphere). Plates were incubated at 37 °C and OD₆₀₀ measured every 8 min for 24 h in a Victor Nivo plate reader (PerkinElmer). For growth curves: to determine alterations to growth dynamics in the knockout strains of the inositol lipid cluster, dense overnight cultures of each BT strain in BHIS were centrifuged at 16,000 × g for 1 min, the supernatant removed and the cells resuspended in an OD-normalized volume of BHIS or BMM before aliquotting 200 μl per well in a sterile 96-well plate (n = 4). For AMP resistance: to identify changes of each strain’s sensitivity to the AMP LL-37, dense overnight cultures of each BT strain were OD-normalized and diluted to a starting OD₆₀₀ of ~0.15 in a total of 200 μl BMM per well, each well supplemented with 0–64.0 μg ml⁻¹ LL-37 (InvivoGen) in duplicate per strain and AMP concentration. Inhibition due to LL-37 was graphically modelled by percent OD at 0 ng ul⁻¹ LL-37: (maximum OD in 24 h − minimum OD in 24 h)/avg(maximum − minimum OD at 0 ng ml⁻¹ per strain) × 100. IC₅₀ was calculated in Prism by fitting ‘concentration inhibitor vs normalized response with variable slope’ and between-strain comparisons using Tukey’s multiple comparisons. The data shown are representative of two experiments.

Post-mortem frozen human brain sections from MS and control donors were obtained from the Rocky Mountain MS Center Tissue Bank (Englewood, CO, USA) after approval by the Medical University of Gdansk (Poland) bioethics committee (NKBBN/253/2018). Lightly frozen 1 cm by 1 cm fragments were dissected from the white matter (WM) and periventricular WM plaques. Corresponding areas of WM were dissected from control brains. The fragments were deep-frozen in liquid nitrogen, ground using mortar and pestle, and homogenized in Fenozol reagent (A&A Biotechnology, 203–50). Total RNA was isolated from approximately 200 mg of homogenized tissue using the Total RNA Mini kit (A&A Biotechnology, 031–100) in an RNAase-free environment according to the manufacturer’s instructions. RNA quantification and quality control were performed spectrophotometrically at 260 nm and 280 nm using a PerkinElmer VICTOR Nivo plate reader (Perkin Elmer, USA) equipped with a μDrop Plate (Thermo Scientific, N12391). Isolated total RNA was stored at -20°C until used.

The endpoint PLA₂ activity assay was run as described previously [23 (link)]. The snake venoms were dissolved at a concentration of 10 mg/mL in assay buffer (10 mM Tris pH 8, 100 mM NaCl, and 10 mM CaCl₂), and a 2-fold serial dilution (10 steps) was prepared. Then, 100 µL/well of each dilution was added to a 96-well plate, together with 100 µL/well of NOB (final concentration 0.25 mM). The plates were shaken at 300 rpm for 2 minutes and then incubated at 37°C for 40 minutes. The plates were then centrifuged (3,000 × g, 4°C, 3 minutes) before the absorbance was recorded at 405 nm using a VICTOR Nivo plate reader (PerkinElmer, Waltham, MA, USA) at 25°C. All reactions were run in duplicates and the absorbance values were shown as averages after subtracting a blank control containing no venom. EC₅₀ values (the venom concentration inducing half of the maximum absorbance at 405 nm proportional to product conversion) were determined using nonlinear fitting with sigmoidal dose–response equation of the venom dose curves analyzed by GraphPad Prism 9 software (GraphPad Software, La Jolla, CA, USA).

CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used and adapted to measure the ATPs released by unstimulated or stimulated cells with DCF001 and/or TNFα. Briefly, 50 µL of the culture medium of each sample was incubated with an equal volume of CellTiter-Glo^® Reagent in the 96-well opaque-walled plate. Luminescence was recorded 10 min after reagent addition using a VICTOR^® Nivo™ Plate Reader (PerkinElmer, Waltham, MA, USA) and reported in relative light units (RLU). Data were expressed as the mean ± SD of four replicates for each sample. In parallel, wells containing medium unconditioned by cells were used to measure background luminescence.

At each time point, a portion of the medium was collected from control and test culture dishes. Freshly prepared media that were not used for cultivation served as negative controls. Human albumin was detected in culture media by the enzyme-linked immunosorbent assay (ELISA) using the Albumin Human ELISA Kit-EHALB (Thermofisher, Waltham, MA, USA) according to the protocol provided by the manufacturer. The analyses were made on a Victor Nivo plate reader (Perkin Elmer, Waltham, MA, USA).