Picoprobe nadph quantitation fluorometric assay kit

The concentrations of cellular pyruvate and NADPH were determined by the Pyruvate Colorimetric/Fluorometric Assay Kit and PicoProbe™ NADPH Quantitation Fluorometric Assay Kit, respectively (K609-100 and K349-100; BioVision, Milpitas, CA, USA). On 6 cm plates, HEK293T (1.5 × 10⁶ cells), MRC-5 (3 × 10⁵ cells), HFL-1 (3 × 10⁵ cells) and H1299 (3 × 10⁵ cells) and MCF-7 (6 × 10⁵ cells) were cultured for 24 h in medium containing 20 mM L-malate at 37 °C with 5% CO₂. Cells were treated with EA or MDSA for 48 h. After harvesting, cells were sonicated in 100 µL phosphate-buffered saline (PBS). After centrifugation, the supernatant was deproteinized using a 10 kDa spin column (Acrodisc® syringe filter, Pall Life Sciences) and analyzed using working mixtures in a 96-well plate with the Biotek® multi-mode microplate reader to detect fluorescence at Ex/Em=535/587 nm (Agilent, Santa Clara, CA, USA).

Levels of NADH, NADPH and OAA in the islets or MIN6 cells were measured with PicoProbe NADH Fluorometric Assay Kit (BioVision), PicoProbe NADPH Quantitation Fluorometric Assay Kit (BioVision) and Oxaloacetate Colorimetric/Fluorometric Assay Kit (BioVision), respectively, according to the manufacturer's instructions. The isolated islets with similar size were used for the above experiments.

Mice were decapitated and 10 mg of cortex tissues of C57BL/6J mice were prepared for the test. Glycogen content was detected using a Glycogen Assay Kit (BioVision, K648, United States). GP activity was analyzed with the Glycogen Phosphorylase Activity Assay Kit (Genmed Scientifics, GMS50092.1, China); pyruvate levels were determined using the Pyruvate Colorimetric/Fluorometric Assay Kit (BioVision, K609, United States); lactate levels were determined using the Lactate Colorimetric Assay Kit II (BioVision, K627, United States); NADPH levels were determined using the PicoProbe™ NADPH Quantitation Fluorometric Assay Kit (BioVision, K349, United States); adenosine triphosphate (ATP) levels were determined using the ATP Colorimetric/Fluorometric Assay Kit (BioVision, K354, United States); and glutamate levels were determined using the Glu Assay Kit (Solarbio, BC1580, China).