A calcium indicator for mitochondria
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
Rhod-2 AM是红色荧光钙指示剂rhod-2的乙酰甲基（AM）酯。AM基团有助于细胞摄取，并由细胞质酯酶去除，导致rhod-2在细胞内积累。Rhod-2 AM选择性地积累在线粒体内，因此通常用于监测该细胞器内的钙变化。激发和发射极大值分别为557和581 nm。
1.Minta, A., Kao, J.P., and Tsien, R.Y.Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophoresJ. Biol. Chem.264(14)8171-8178(1989) 2.Jean-Quartier, C., Bondarenko, A.I., Alam, M.R., et al.Studying mitochondrial Ca2+ uptake - a revisitMol. Cell. Endocrinol.353(1-2)114-127(2012) 3.Paredes, R.M., Etzler, J.C., Watts, L.T., et al.Chemical calcium indicatorsMethods46(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 reperfusionCell Calcium51(6)445-451(2012) 5.Bodnár, D., Geyer, N., Ruzsnavszky, O., et al.Hypermuscular mice with mutation in the myostatin gene display altered calcium signallingJ. Physiol.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 cellsAm. J. Physiol. Gastrointest. Liver Physiol.307(3)G286-G294(2014)
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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°C in 4% paraformaldehyde. Cells are washed in PBS, and images are acquired with Zeiss Apotome and Axiovision software[1]. |
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参考文献: [1]. Brisac C, et al. Calcium flux between the endoplasmic reticulum and mitochondrion contributes to poliovirus-induced apoptosis. J Virol. 2010 Dec;84(23):12226-35. |
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