Multimode plate reader

The method described by Spinazzi et al.^{44 (link)} was used to measure CS activity. The tissues were crushed in homogenate buffer (sucrose/Tris/MgCl/015-21274/207-06275/132-001751, Fujifilm Wako Pure Chemicals Corp.) using a biomasher and further crushed using a sonicator. The tissue was then centrifuged at 500×g for 5 min at 4 °C and the supernatant was collected. The protein concentration of the collected supernatant was determined using the BCA method (297-73101, Fujifilm Wako Pure Chemicals Corp.). 5 μL of sample solution was diluted to an equal volume (1 mg/mL) of protein and 50 µL of 1 mM DTNB (043-16403, Fujifilm Wako Pure Chemicals Corp.), 10 µL of 3 mM acetyl CoA (00546-96, Nacalai Tesque, Kyoto, Japan), 9 µL of 10% TritonX-100, 1 µL of 1 M Tris–HCl (pH 8.0), and 20 µL of 10 mM oxaloacetic acid (00546-96, Nacalai Tesque) were added. After mixing, changes in absorbance at 412 nm were immediately measured using a multimode plate reader (Tecan, Menendorf, Switzerland) at 37 °C for 15 min at 5 s intervals. The results were analyzed using SparkControl Magellan 2.2, and CS activity was evaluated by analyzing five consecutive changes in absorbance with the largest slope per minute.

To assess metabolic activity, 1 × 10⁴ cells were plated in 96-well culture plates (Nunc) in complete DMEM. Sixteen hours later, cells were challenged with 30, 60, and 120 s of plasma treatment before further incubation for 6 h or 24 h. Subsequently, wells were loaded with 100 μM of resazurin (Alfa Aesar) that is transformed to fluorescent resorufin by metabolically active cells. The plate was incubated for 2 h at 37 °C, and fluorescence was measured using a multimode plate reader (Tecan) at λ_ex 535 nm and λ_em 590 nm. Metabolic activity was shown as percent of the untreated control. To determine toxicity, cells were loaded with sytox orange (1 μM; Thermo Fisher) for 30 min at 37 °C. Cells were imaged with a 20x objective using a live cell high throughput imaging system (Operetta CLS; PerkinElmer). Algorithm-based quantitative image analysis was performed using dedicated software (Harmony 4.8; PerkinElmer).

Primary keratinocytes were seeded at 70–80% confluence in black 96 well flat bottom plate (Corning) and incubated with CaCl₂ for different time intervals or treated with Tg or Torin 1 or EGTA (as control) in combination with CaCl₂ for 6 h or 48 h. Intracellular free calcium was measured using Fluo-4 NW calcium assay kit (Molecular probes, F36206). Briefly, Fluo-4 NW dye was added to the cells 2–6 h prior to the end of time point. The total cellular fluorescence was measured at 516 nm with an excitation of 494 nm using Tecan multi-mode plate reader. The emission fluorescence intensity values were normalized with the cell numbers measured by Resazurin (Sigma-Aldrich)-based cell viability assay. The fold change in fluorescence intensity between the treatment and control was measured and then plotted.

Freshly isolated mitochondria (20 μg) were incubated in mitochondrial respiration buffer and 50 μmol/L 2′7′ dichlorofluorescin (DCF). ROS emission was measured under state III respiratory condition through the addition of 10 mmol/L Succinate and 1 μmol/L Rotenone, and 2.5 mmol/L ADP. Relative fluorescence change (Ex: 480 nm/ Em: 520 nm) was measured using a Tecan multimode plate reader.

CCK-8 assay was performed to investigate cell viability [29 (link)] (Cat#: K1018; APExBIO, China). Briefly, KYSE450 and KYSE510 cells (3 × 10³ cells) were seeded in a 96-well plate. At the indicated times, CCK-8 (10 μL) reagent was added to the wells with cells, and the cells were incubated further for 2 h. Finally, the optical density (OD) was measured at 450 nm with a multimode-plate-reader (Tecan, Switzerland).

1 × 10⁴ cells were challenged with ADDA5, KCN or NaN₃ in the presence or absence of PTM for 3 and 24 hours. Subsequently, wells were loaded with 100 µM of resazurin (Alfa Aesar) that is transformed to fluorescent resorufin by metabolically active cells. The plate was incubated for 2 h at 37 °C. Fluorescence was measured in multimode plate reader (Tecan) at λ_ex 535 nm and λ_em 590 nm and normalized to untreated control. Four hours after plasma treatment, apoptosis was assessed by staining with cell event™ caspase 3/7 (ThermoFisher) for 30 min at 37 °C. Subsequently, cells were detached using accutase (BioLegend), and accutase containing 4′,6-diamidino-2-phenylindole (DAPI; BioLegend) was added to label terminally dead cells. Cells were subjected to flow cytometric analysis (CytoFlex; Beckman-Coulter). At least 3000 cells were acquired in the caspase⁻/DAPI⁻ gating region. Data analysis was performed utilizing Kaluza 1.5a software (Beckman-Coulter).