Cell experiment:

For flow cytofluorometry, cells are harvested, pelleted, and resuspended in ice-cold PBS containing 10 mM glucose, 10% fetal bovine serum (FBS), and 10 μM Rhod-2 AM (Rhod2-AM). Mitochondrial calcium levels are determined by the flow cytofluorometry analysis of aliquots of 4×105 cells. For fluorescence microscopy, IMR5 cells are grown on polylysine-coated (10 μg/mL) slides and stained with 7.5 μM Rhod-2 AM in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS for 2 h before poliovirus (PV) infection. Cells are fixed by incubation for 15 min at 4℃ in 4% paraformaldehyde. Cells are washed in PBS, and images are acquired with Zeiss Apotome and Axiovision software[1].

Rhod-2 AM is an acetoxymethyl (AM) ester of the red fluorescent calcium indicator rhod-2. The AM group facilitates cellular uptake and is removed by cytoplasmic esterases, resulting in intracellular accumulation of rhod-2.[1],[2],[3] Rhod-2 AM selectively accumulates within mitochondria and, as a result, is commonly used to monitor calcium changes within this organelle.[4],[5],[6] Excitation and emission maxima are 557 and 581 nm, respectively.[3]

Reference:
[1]. Minta, A., Kao, J.P., and Tsien, R.Y. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. The Journal of Biological Chemisty 264(14), 8171-8178 (1989).
[2]. Jean-Quartier, C., Bondarenko, A.I., Alam, M.R., et al. Studying mitochondrial Ca2+ uptake - a revisit. Molecular and Cellular Endocrinology 353(1-2), 114-127 (2012).
[3]. Paredes, R.M., Etzler, J.C., Watts, L.T., et al. Chemical calcium indicators. Methods 46(3), 143-151 (2008).
[4]. Wang, S., Chen, J., and Valderrábano, M. Nutrient restriction preserves calcium cycling and mitochondrial function in cardiac myocytes during ischemia and reperfusion. Cell Calcium 51(6), 445-451 (2012).
[5]. Bodnár, D., Geyer, N., Ruzsnavszky, O., et al. Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling. Journal of Physiology 592(6), 1353-1365 (2014).
[6]. Elamin, E., Masclee, A., Troost, F., et al. Cytotoxicity and metabolic stress induced by acetaldehyde in human intestinal LS174T goblet-like cells. American Journal of Physiology and Gastrointestinal Liver Physiology 307(3), G286-G294 (2014).