Sparkcontrol magellan

The XTT assay (2,3-Bis-2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide salt, Sigma-Aldrich; Saint Louis, MO, USA) is a colorimetric test based on the reduction of tetrazolium salts into an orange formazan dye. The reduction occurs only in metabolically active cells. In order to carry out this test, 20,000 eACs/cm² were seeded on a 96-well plate in the presence of HG-DMEM medium supplemented with 10% FCS and PSA. At 80% of confluency, treatments were added and the cells were incubated at 37 °C, 5% CO₂ for 72 h. Then the cells were incubated in the presence of a mixture containing XTT. Absorbance measurements were made at 450 nm for samples and background noise at 600 nm, the difference between these two measurements determines the metabolic activity. Measurements were made with a microplate reader (Spark control Magellan, TECAN^®, Lyon, France). All experiments were performed in triplicate and repeated 4 times.

The metabolic activity was analyzed using the XTT test directly in the plates used for the cytotoxicity experiments. This assay is based on the reduction of tetrazolium salt to orange-colored formazan only by metabolically active cells. After each time of treatment, an XTT assay was performed according to the manufacturer’s instructions (Roche, Bale, Switzerland). Optical density (OD) measurements were taken at 490 and 650 nm with a microplate reader (Spark control Magellan, TECAN).

To assay for the minimum [BAPTA] to be used in experiments for calcium chelation, varying BAPTA concentrations were prepared in reconstitution buffer (25 mM HEPES-KOH pH-7.4, 100 mM KCl). The calcium indicator Fluo-4 was then mixed with the samples, and its fluorescence (Ex: 494 nm and Em: 516 nM) was recorded using the Tecan SPARKcontrol Magellan microplate reader.

The viability of SH-SY5Y cells was analyzed using the WST-1 colorimetric assay (Cat No. 11644807001, Roche Diagnostics GmBH, Mannheim, Germany). Cells were seeded in transparent flat-bottom 96-well plate at a density of 18,000 cells per well and after 48 h the cells were treated with increasing concentrations of EGCG for a duration of 24 h. Following this period, 10 μL of WST-1 was added to each well and after one hour of incubation the absorbance was measured in a Tecan Spark 20 M multiplate reader (Männedorf, Switzerland) at 450 nm and 650 nm (as reference wavelength and subtracted during analysis) employing Tecan’s SparkControl magellan version 1.2. The cell viability experiment was performed in three independent biological experiments with four replicates per repeat. Results were normalized over the 0 μM EGCG non-treated condition and statistical analysis was performed by one-way ANOVA, Dunnett´s Multiple Comparison Test, by use of GraphPad Prism version 5.0.4 (GraphPad Software Inc., San Diego, CA, USA). p = < 0.05 (*p < 0.05); p = < 0.005 (***p < 0.005). Results are displayed as mean±S.D. from n = 3 biologically independent experiments.

Two different in vitro luciferase assays were performed in the study. If cells need to be kept alive for continuous tracing of luciferase expression, 150 µg/mL D-luciferin was directly added to the medium, and the cells were incubated at 37°C for 10 min. The bioluminescent signals were collected using an IVIS Imaging System (Xenogen) following the manufacturer’s instructions (24 (link)). The end-point luciferase assay will be performed according to the manufacturer’s instructions (Promega) if the cells need not be kept alive. Cells were plated and cultured in white-flat 96-well cell culture plates before the infection of luciferase-tagged viruses. The medium was removed at detection, and 25 µL lysis buffer was added to each well. The plates were vortexed on a plate vortexer for 10 min to lyse the cells, and 100 µL luciferase assay reagent was added to each well. The bioluminescent signals were collected with a SparkControl Magellan plate reader (Tecan, Männedorf, Switzerland).
An in vivo luciferase assay was performed to quantitatively trace the dissemination of MCMV-Luc in mice (24 (link)). MCMV-Luc-infected mice were injected intraperitoneally (i.p.) with 1.5 mg D-luciferin, and the mice were anesthetized with isoflurane inhalation. The bioluminescent signals were recorded 10 min post-D-luciferin injection using the IVIS imaging systems.

A glutamate release assay using ND5_S (with varying lipid compositions as indicated in Fig S9) and glutamate-entrapped t-lipos was performed as described previously (Bao et al, 2016 (link); Nellikka et al, 2021 (link)). Here, 4 nM t-lipo was mixed with 150 nM NDs (as indicated) in a 96-well flat black plate with 1 μM iGluSnFR in the reaction buffer (25 mM Hepes, pH-7.4, 100 mM KCl, and 2 mM β-mercaptoethanol). Glutamate release from t-lipo was measured as an increase in iGluSnFR fluorescence in a SparkControl Magellan microplate reader (Tecan). The following parameters were set for the experiment in the plate reader: (1) excitation and emission wavelengths were set to 480 and 510 nm, respectively; (2) temperature: 30°C. Maximum glutamate release percentage was recorded by adding 5 μl of 5% Triton, and the iGluSnFR fluorescence was recorded for an additional 10 min. The raw fluorescence values were converted into % glutamate release and plotted as a function of time.

YPL (1% yeast extract, 2% peptone, 2% lactate), YhPL (0.5% yeast extract, 2% peptone, 2% lactate), YPD (1% yeast extract, 2% peptone, 2% glucose), SL (0.67% yeast nitrogen base without amino acids, 2% lactate), YPD (1% yeast extract, 2% peptone, 2% glucose), SD-N (0.17% yeast nitrogen base without amino acids and ammonium sulfate). YhPL was used only for GFP cleavage autophagy assay. Growth curves were performed using 96-well plates and continuous OD₆₀₀ measurements using a Tecan SparkControl Magellan plate reader. Yeast were grown at 30°C.