Spark 10m plate reader

The transport of Ca²⁺ across the inner mitochondrial membrane was monitored spectrophotometrically with an arsenazo III indicator at 675–685 nm using a Tecan Spark 10M plate reader (Tecan Group Ltd, Männedorf, Switzerland) as previously described [39 (link)]. Skeletal muscle mitochondria (~0.25 mg of mitochondrial protein/mL) were suspended in an incubation medium containing 150 mM sucrose, 50 mM KCl, 2 mM KH₂PO₄, 10 μM EGTA, and 10 mM HEPES-KOH (pH = 7.4) and were energized with 2.5 mM glutamate + 2.5 mM malate. To determine the ability of the mitochondria to retain Ca²⁺, 10 μM CaCl₂ was added into the reaction medium successively, with an interval of about 90 s. After several additions, external (Ca²⁺) increased, indicating a massive release of the ion from the organelles due to the opening of the MPT pore in the inner mitochondrial membrane. The total amount of the added Ca²⁺ ions that induced their spontaneous release from mitochondria due to the induction of the MPT pore opening was interpreted as the calcium retention capacity of the organelles.

Strains were grown overnight at 30 °C in 5 mL SD −ADE medium. For spotting assay, cells were diluted to OD₆₀₀ = 0.6 and were then five-fold serially diluted and spotted on SD media containing 2 mg/L adenine and the indicated concentrations of the chemical chaperones. Plates were incubated at 30 °C for 2 days. For kinetic growth assays, cells were diluted to OD₆₀₀ = 0.01 in a final volume of 200 µL in a 96-well microplate (Greiner Bio-One, Kremsmünster, Austria). The plate was incubated at 30 °C with continuous shaking and OD₆₀₀ was measured using a Tecan SPARK 10M plate reader (Tecan Trading AG, Männedorf, Switzerland). Assays were performed in triplicates and the results displayed are representative of three independent biological repeats.

Adenine (6 mg/mL) was dissolved in PBS by heating to 90 °C for 3 h to obtain a monomeric solution. The adenine solution was then mixed with DMSO or DCA at the indicated concentrations in a black clear flat-bottom 96-well microplate (Greiner Bio-One, Kremsmünster, Austria). The samples were mixed with ProteoStat (prepared according to the manufacturer’s instructions) at a 1:100 ratio. Adenine diluted in PBS was used as a control for DMSO-treated samples, while 0.25% DMSO was supplemented as a control for DCA assays. Fluorescence was measured over time using a Tecan SPARK 10M plate reader (Tecan Trading AG, Männedorf, Switzerland) with excitation and emission wavelengths of 485 nm and 620 nm, respectively. Assays were performed in triplicates, and the results displayed are representative of three independent repeats.

Approximately 3000–4000 cells/well were plated overnight in 96-well flat-bottomed plates. The next day, cells were exposed to either afatinib or crizotinib (Sellekchem) alone or in combination for 72 h after which MTS solution (Promega, Madison, WI, USA) was added and absorbance at 490 nM was measured using Tecan Spark 10 M plate reader (Tecan Trading AG, Switzerland), to determine the inhibitory concentration of the drugs according to the manufacturer’s protocol. CDI was calculated as CDI = AB/(A × B). According to the absorbance of each group, AB is the ratio of the combination groups to DMSO group; A or B is the ratio of the single-agent group to the DMSO group.

Mtb Lux was grown to log phase (kanamycin 25 μg/mL) and diluted in 30 mL inkwells (Corning Lifesciences) to an OD₆₀₀ = 0.05 in 7H9 + OADC + 5 μg/mL clavulanate with varying concentrations of amoxicillin, meropenem, or both. 100 μL of these cultures were pipetted in duplicate into a white 96-well polystyrene plate (Greiner Bio-One) and luminescence was measured in a Synergy H1 microplate reader from BioTek Instruments, Inc. using the Gen5 Software (2.02.11 Installation version). The correlation between relative light units (RLU) and CFU is shown in Msm in Figure 5—figure supplement 1.
Msm Lux was grown to log phase and diluted into white 96-well polystyrene plates to an OD₆₀₀ = 0.05. Plates were sealed with 4titude Moisture Barrier Seals and shaken continuously at 37°C. Luminescence measurements (RLU) were taken at 15-min intervals integrated over 1000 ms in a TECAN Spark 10M plate reader for 18 hr.

HCT116 and HEK293T cells were seeded in tissue-culture-treated 96-well plates at a density of 3 × 10⁴ cells per well and transfected with siRNAs at a final concentration of 10 or 20 nM, using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Göteborg, Sweden), as described in “Cell culture and siRNA transfection”. After 24 h, cells were transfected with a β-catenin/TCF luciferase reporter (Super 8× TOPflash; a gift from Randall Moon, Addgene plasmid #12456) and a Renilla control plasmid (pIS1; a gift from David Bartel, Addgene plasmid #12179), using Lipofectamine 2000. Cells were treated after 3 to 6 h with Wnt agonist Wnt3a or controls. After 24 h, cells were lysed using a homemade dual luciferase assay, following a protocol described in [21 (link)]. Luminescence was measured using the Spark 10 M plate reader (Tecan), and TOPflash values were normalized to Renilla activity.

Steady-state ultraviolet–visible (UV–Vis) absorption spectra were recorded using a Cary 300 UV–Vis spectrometer (Agilent Technologies), equipped with a Versa20 Peltier-based temperature-controlled cuvette chamber (Quantum Northwest), and fluorescence data were recorded using a LPS 220 spectrofluorometer (PTI, Monmouth Junction, NJ, USA), equipped with a TLC50TM Legacy/PTI Peltier-based temperature-controlled cuvette chamber (Quantum Northwest).
Thermodynamic dissociation constants for NFAST:CFASTn (n = 8–11) couples were determined using peptides synthesized for CFASTn (n = 8–11) and recombinantly purified NFAST. The affinity for NFAST:CFAST11 in the presence of 10 µM HMBR was determined independently from a minimum of three different purifications of NFAST. NFAST:CFAST11 was then run in parallel as an internal control for the determination of the other NFAST-CFAST combinations, which were all performed on the same day with the same preparation of NFAST. Thermodynamic dissociation constants were determined with a Spark 10M plate reader (Tecan) and fit in Prism 6 to a one-site-specific binding model.