Infinite f200 pro spectrophotometer

Ellman’s Reagent [5,5′-dithiobis-(2-nitrobenzoic acid) or DNTB] was used to measure the intracellular content of GSH. DTNB reacts with GSH, producing 2-nitro-5-thiobenzoic acid and glutathione disulfide, a yellow-colored product measured at 405 nM [26 (link)] using an infinite^® F200 PRO spectrophotometer (Tecan, Männedorf, Switzerland). After treatment, cells were harvested, washed in 1× phosphate-buffered saline (PBS), and lysed with CelLytic^TM reagent (Merck Millipore, Darmstadt, Germany). Protein concentration was measured using the Bradford assay with an infinite^® F200 PRO plate reader (Tecan, Männedorf, Switzerland) and extrapolated using the standard curve obtained from bovine serum albumin (BSA). An equal protein amount (minimum 20 μg) for each sample was used to measure GSH content. Cell lysates were deproteinized using trichloroacetic acid (TCA) and then centrifuged at 14,000 rounds per minute (RPM) for 5 min to remove the precipitated protein. 50 μL of supernatant was added to Tris-EDTA pH 8.9 and mixed with DTNB (0.01 M in methanol). GSH content was measured spectrophotometrically at 405 nm and expressed as fold change in treated cells compared to untreated cells. N-acetyl cysteine (NAC, 5 mM) was used as an internal control for the assay.

CPDs were measured by ELISA. Epidermal sheets were separated by NH₄SCN as above. Epidermal DNA was isolated using a DNeasy Blood and Tissue kit (Qiagen) and denatured at 100 °C for 10 min. DNA (10 ng) was dried onto protamine sulfate-coated 96-well plates (Nunc), and ELISAs were performed using anti-CPDs (Cosmo Bio), biotinylated goat anti-mouse IgG (Invitrogen), streptavidin–horseradish peroxidase (R&D) and a TMB Substrate Reagent set (BD), as per the anti-CPDs manufacturer’s protocol. Absorbance was measured using an Infinite F200 Pro spectrophotometer (Tecan).

Jurkat cells (4.5 × 10⁶/3 ml) were treated with 500 μM for D-dA or dA or 100 μM for D-dT or dT for 24 h. Cells were collected by centrifugation for 7 min at 130 g, washed in PBS, and their DNA was extracted with QIAamp DNA Mini Kit (QIAGEN), according to the manufacturer's instructions. DNA extracted was sublimated under vacuum in Savant Speed VAC SC110 Concentrator Centrifuge (Savant) to remove ethanol contamination, quantified by Tecan Infinite f200 PRO spectrophotometer (Tecan), and digested according to Quinlivan and Gregory [14 (link)]. Briefly, 40 μg of DNA/2 ml H₂O was treated with 100 U benzonase (DBA Italy s.r.l., Milan, Italy), 120 mU phosphodiesterase I (DBA Italy s.r.l.), and 80 U alkaline phosphatase (Sigma-Aldrich) in 2 ml Tris-HCl buffer (20 mM Tris-HCl, 100 mM NaCl, 20 mM MgCl₂, pH 7.9; all obtained from Sigma-Aldrich) o.n. at 37°C. The resulting deoxynucleosides were subsequently analysed in NMR as described below.