Calcium Orange AM

Calcium Orange AM is an intracellular calcium reporter. Specific fluorescence can be detected when free calcium binds to Calcium Orange AM (Ex/Em=549/576 nm). Calcium Orange AM does not enter the vacuoles and does not compartmentalize into acidic vesicles[1].

Research Areas >> Others

Signaling Pathways >> Others >> Others

Guidelines[2] (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs). 1. Calcium Orange AM is freshly prepared in dehydrated DMSO before each experiment. 2. Prepare the recording medium for measuring [Ca2+]i in cells consisted of 20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, and 13.8 mM D-glucose adjusted to pH 7.4 by adding 1 M KOH. 3. Exchange the culture medium for 1 mL of a recording medium including 10 μM Calcium Orange AM. 4. Incubate cells under conditions of 5% CO2 and 37°C for 40 min. 5. Remove the recording medium, including calcium orange AM from the dish, and add the culture medium after washing it three times with 1 mL of the recording medium. 6. Incubate the cells for 1 h. After incubation, exchange the culture medium for the recording medium again. 7. Detect fluorescence.

[1]. Lam CM, et al. Monitoring cytosolic calcium in the dinoflagellate Crypthecodinium cohnii with calcium orange-AM. Plant Cell Physiol. 2005 Jun;46(6):1021-7.

[2]. Inami W, et al. Intracellular calcium ion concentration measurement using a phase-modulation fluorescence lifetime method with compensation for phase shift due to the presence of proteins. Anal Sci. 2013;29(2):199-203.