Victor 2 reader

Human colonic carcinoma (HCT116) and human embryonic kidney (HEK293a) cells were from the European and American Collections of Cell Cultures. HCT116 cells were maintained in McCoy’s 5A medium supplemented with 10% foetal bovine albumin (FBS), 2 mM L-glutamine, 100 U/ml penicillin / 100 µg/ml streptomycin (P/S) and 10 mM HEPES, pH 7.2 (complete McCoy). HEK293a cells were maintained in DMEM supplemented with 10% FBS, P/S and 10 mM HEPES (complete DMEM), both in humidified 5% CO₂ / 95% air, at 37 °C.
For the probe toxicity testing, cells were seeded on standard 96-well plates pre-coated with collagen IV at 2.5 × 10⁴ cells/well, grown for 24 h, and then exposed to NanO2-IR at 1 mg/ml in complete DMEM for different periods. After that, total cellular ATP levels were measured using CellTiter-Glo Kit, white 96-well plates (Greiner Bio One) and Victor 2 reader (PerkinElmer) under luminometry settings^{45 (link)}.
In the oxygenation experiments, HCT116 cells grown to 90–100% confluence were trypsinised, washed with DMEM, counted and transferred into 0.5 ml qPCR tubes at 0–5 × 10⁶ cells per tube. Then the cells were precipitated to the bottom by mild centrifugation at 200 g, old media was aspirated, fresh complete DMEM with 1 mg/ml NanO2-IR was added to each tube, cells were re-suspended and precipitated as above. After 30 min incubation at 37 °C tubes with cells were imaged.

Oxidative stress was measured using the fluorescent probe 2, 7-dichlorofluorescein diacetate (DCF) (Sigma-Aldrich) in INS-1E cells seeded in black 96-well plates. After treatment, cells were loaded with 10 μM DCF for 30 minutes at 37°C and washed. DCF fluorescence was quantified in Victor 2 reader (Perkin Elmer, Germany). Cells were then lysed and total protein measured. Data are expressed as DCF fluorescence corrected by total protein.

Protein levels of galectin-1, galectin-3, IL-8, RANTES, and MIP-3α were simultaneously quantified using a custom-designed multiplex electrochemiluminescence (ECL) immunoassay, Sector Imager 2400 and Discovery Workbench Software (Meso Scale Discovery, Gaithersburg, MD), validated against traditional ELISA (9 (link), 37 (link), 38 (link)). Galectin-1 and -3 levels were also assessed by traditional commercially available ELISA (R&D Systems). The concentrations of soluble extracellular mediators released in the cell culture supernatants were measured in picograms/ml. The galectin levels in cell lysates (cell-bound galectins) were measured also in picograms/ml by ELISA and then normalized to the total protein of cell lysate, thus quantifying galectins in nanograms/mg total protein. Total protein levels were measured using the Pierce BCA protein assay. The ELISA and the BCA assays were read using a Victor2 reader (PerkinElmer Life Sciences).