Glass bottomed culture dishes for imaging

Dulbecco's Modified Eagle Medium (DMEM), DMEM calcium-free media, ATP, and Fluo-4 AM were purchased from Gibco/Thermofisher (Waltham, MA, USA). CyQUANT™ MTT Cell Proliferation Assay Kit was ordered from Invitrogen (St. Louis, MO, USA). Glass-bottomed culture dishes for imaging from MatTek (Ashland, MA). Real-time PCR primers were ordered from Gene Link (NY, USA), cDNA synthesis and realtime PCR kits were from Solis BioDyne (Tartu, Estonia). Cell culture and treatment Human proximal kidney (HK-2) cells obtained from Applied Biological Materials Inc, Canada, were maintained in Dulbecco's Modified Eagle Medium (Gibco/ Thermofisher) with high glucose (4.5 g/L) supplemented with 10% FBS, 2 mM L-glutamine, and 1 % penicillin-streptomycin at 37°C with 5% carbon dioxide. The standard DMEM media contains 1.8 mM calcium as reported in the company datasheet. Adjusting the required calcium concentration was done in calcium-free media from the same company by adding CaCl2. Cells were seeded overnight and left to reach ∼ 60-70% confluency before treatments. The next day, media was removed and replaced either by standard media or calcium-free supplemented with CaCl2 to the required concentration, and the cells were grown for an additional 18-20 h before processing. ATP was added to the media to a final concentration of 50 μM.