Biotek multi mode microplate reader

NAD⁺ and NADH levels were determined using the NAD/NADH-Glo^TM Assay (G9071; Promega, Madison, WI, USA). On 6 cm plates, HEK293T (1.5 × 10⁶ cells), HFL-1 (3 × 10⁵ cells), and MRC-5 (3 × 10⁵ cells) were treated for 48 h with 25 µM EA or MDSA at 37 °C with 5% CO₂. HEK293T (2 × 10⁶ cells in 100 µL PBS), HFL-1 and MRC-5 (2 × 10⁵ cells in 100 µL PBS) were sonicated, and the supernatants were deproteinized using the 10 kDa spin column (Acrodisc® syringe filter, Pall Life Sciences). The supernatants (2 × 10⁴ cells in 100 µL PBS) were mixed with an equal volume of detection reagent in a 96-well plate, and the levels of NAD⁺ and NADH were determined using luminescent signals from the Biotek® multimode microplate reader (Agilent, Santa Clara, CA, USA).

Utilizing the Glutamine/Glutamate-Glo™ Assay kit (#Cat. J8021; Promega, Madison, WI, USA), the levels of glutamate were measured. On 6-cm dishes, cells were treated with 150 µM EA at 37 °C, 5% CO₂ and 5% humidity. After 48 h of treatment, cells were washed twice with 200 µL PBS, resuspended in 30 µL PBS, and then combined with 15 µL Inactivation Solution I. (0.3 N HCl). After 5 min of shaking, 15 µL of Tris Solution I (450 mM Tris-HCl, pH 8.0) was added to each sample and mixed for 1 min. Using the 10 kDa spin column (Nanosep, Acrodisc® syringe filter, Pall Life Sciences, NY, USA), the samples were deproteinized, and 50 µL of each sample was transferred into a white 96-well plate. In each well, 50 µL of Glutamate Detection Reagent was added and mixed with the sample by shaking the plate for 1 min. After incubating for 60 min at room temperature, the levels of glutamate were measured using the BioTek® multimode microplate reader to detect luminescent signals (Agilent, Santa Clara, CA, USA).

The ATP content of cells was determined using a CellTiter-Glo® 2.0 Cell Viability Assay (#Cat. G9242; Promega, Madison, WI, USA). On 6 cm plates, the cells (5 × 10⁵/ml) were re-suspended in 100 µL PBS and reacted with an equal volume of the assay buffer. The luminescence was detected using the Biotek® multi-mode microplate reader (Agilent, Santa Clara, CA, USA). The ATP content was also detected using a BioTracker™ ATP-Red Live cell dye (#SCT-045, Sigma-Aldrich, St. Louis, MO, USA) was used to measure ATP levels in the cell. The cells (5 × 10⁵/ml) were washed in 200 µL phosphate-buffered saline (PBS) buffer and centrifuged for 5 min at 300xg. The cells were resuspended in 99.9 µL PBS buffer with 0.1 µL ATP probe (10 mM), and a 37 °C humidified incubator was used to incubate the cells for 30 min. Finally, the Cells were washed with PBS buffer and subjected to fluorescence-activated cell sorter (FACS) analysis. Using a BD Accuri C6 Plus flow cytometer, data was collected (BD Biosciences, San Jose, CA, USA).