Infinite m1000 pro plate reader

The optimal concentration of ASMDM-1 in BEAS-2B was determined using previously detailed methods [25 (link)] with modifications. Briefly, BEAS-2B cells were seeded in 24-well plates (Corning Corp, New York, NY, USA) at 5 × 10⁴ cells/mL. The plates were incubated at 37 °C (5% v/v CO₂) until confluent. Following incubation, the media was discarded, and cells were washed once with 1 × PBS. DMEM or diluted ASMDM-1 was then added to the cells at varying concentrations; 0-, 20-, 40-, 60-, and 80% or 100% ASMDM-1. Following incubation, the supernatant was removed from all wells and plates and washed with 1 × PBS.
To assess cell viability, cells were detached from plates by washing with 1 × trypsin-EDTA at 37 °C for 10 min. Cell density and viability (% live cells) was measured using trypan blue staining using a hemocytometer (Neubauer, Stallikon, Switzerland). To assess cell metabolic viability, 0.05% w/v resazurin solution (Sigma-Aldrich, Sydney) was added to adherent cells, and incubated further at 37 °C with orbital shaking (150 RPM). After 24 h, the fluorescence intensity of the BEAS-2B cells were determined at Ex_544nm and Em_590nm using the Tecan infinite M1000 pro plate reader (Tecan, Grödig, Austria).

ATP amount was measured in C6 cell-cultured medium, in PB serum of experimental animals, and in tumor tissue. After C6 cell treatment with 100 μM BzATP for 24 h, conditioned medium was collected and proceeded as described below. Tumor samples were homogenized in buffer A (25 mM Tris–HCl, 150 mM KCl, 2 mM EDTA, 0.1% EDTA, 10 mM KH₂PO₄, 0.1 mM MgCl₂, pH 7.4) in a mass ratio 1:9. ATP extraction from conditioned medium, PB serum, and tumor homogenates was performed using 1.5% TCA (Sigma Aldrich, USA). Next, pH was adjusted to 7.5–7.8 using 1 M Tris-OH buffer pH 10. For ATP measurement, 1 μl of medium, serum, or clarified tumor homogenate was diluted in 999 μl of buffer A. In total, 100 μl of sample was used for each assay. All samples were diluted to the same final concentration within each type of experiment. The measurement procedures were performed according to the manufacturer’s protocol using ENLITEN® ATP Assay System (Promega, USA). ATP levels were evaluated by luminescence measurement using the Infinite M1000 PRO plate reader (Tecan Trading AG, Switzerland) and expressed in relative light units (RLU).

SOD activity measurement was performed using Superoxide Dismutase (SOD) Activity Assay Kit (BioVision, USA). Ten milligrams of tumor fragments was weighed, homogenized in ice-cold 0.1 M Tris–HCl, pH 7.4 containing 0.5% Triton X-100, 10 mM DTT, and 0.1 mg/ml PMSF, and centrifuged at 12,000 × g for 10 min at 4 °C to discard the cell debris. SOD activity measurement and SOD activity calculation were performed according to the manufacturer’s protocol. The absorbance was measured at 450 nm using Infinite M1000 PRO plate reader (Tecan Trading AG, Switzerland).

Every 10 mg of tumor tissue was disintegrated in 250 μl ice-cold 100 mM phosphate buffer, pH 7. Then, 250 μl of 1.5% TCA was added, incubated for 10 min, and the samples were centrifuged at 12,000 × g for 10 min at 4 °C. Reaction mixture was prepared according to the protocol of Glutathione Colorimetric Detection Kit (Invitrogen, USA). The absorbance was measured at 405 nm using Infinite M1000 PRO plate reader (Tecan Trading AG, Switzerland). The GSSG/GSH ratio was calculated using the following equations: $$\begin{array}{c}ratioGSSG/GS{H}_{\mathit{control}}=\left(2,·,g,s,s,g_{\mathit{contr}},/,\left(g,s,h_{\mathit{contr}},+,\left(2,·,g,s,s,g_{\mathit{contr}}\right)\right)\right)·100\%\\ ratioGSSG/GS{H}_{\mathit{exp}}=\left(2,·,g,s,s,g_{\mathit{exp}},/,\left(g,s,h_{\mathit{exp}},+,\left(2,·,g,s,s,g_{\mathit{exp}}\right)\right)\right)·100\%\end{array}$$