Ez atp assay kit

Epithelial lung fibroblast cells (MRC-5) and airway epithelial cells (Calu-3) [20 ] [21 (link)] were purchased from Korean Cell Line Bank (Seoul, Korea), and HCoV-229E was obtained from ATCC, Manassas, Virginia, USA. The EZ-ATP assay kit (DoGenBio, Korea) was used to measure the total ATP levels, and the alamarBlue reagent (Invitrogen, USA) was utilized to detect cellular viability. Experiments were accomplished as per the manufacturer's guidelines. Hydroxy chloroquine diphosphate (HCQ; positive control) was obtained from Merck (Yongin, Korea). The main stock of HCQ was reconstituted in DMSO and kept frozen (-20 °C) for further treatments. 2-DG was obtained from Merck (Yongin, Korea). 2-DG was first prepared in phosphate-buffered saline prior to each application and then incubated in the virus culture solution according to the experimental conditions. HCoV-S protein-specific antibodies were purchased from Sino-Biologicals (USA). GLUT1, GLUT3, and GLUT4-specific antibodies were purchased from Abcam, USA. Antibodies specific to ABCC9 (Aviva System Biology, USA), IRF1 (Cell Signaling Technology, USA), and FRA-1 (i.e., FOSL1) Santa Cruz Biotechnology, USA, were purchased as mentioned.

The effects of waterlogging stress on photosynthesis were determined by analyzing chlorophyll fluorescence using a pulsed modular fluorometer (FluorPen FP110). Fully expanded leaves were grounded, and chlorophyll (Chl) was extracted in 95% ethanol at 80 °C for 30 min, and the concentrations of Chl a, Chl b, and Chl a + b were calculated according to Czyczyło-Mysza et al. (2013 (link)).
The proline contents in leaves and roots were quantified using the acid ninhydrin method described by Eom et al. (2022 (link)), calculated based on a calibration curve derived from standard proline solutions, and expressed as ng/mg of fresh weight (FW).
The total content of ATP was analyzed using an EZ-ATP Assay Kit (DoGenBio Co., Seoul, Korea) according to the manufacturer’s instructions, and measured using a fluorescence microplate reader equipped with a 535 nm excitation filter and a 595 nm emission filter.