Calcium Indicators

Calcium flux assays are widely used for in-cell measurement of agonist-stimulated and antagonist-inhibited signaling through G protein–coupled receptors (GPCRs), a large and active target class relevant in drug discovery. In neurons, intracellular calcium plays a critical role in induction of synaptic activity and activation of signaling pathways. Functional imaging of calcium as a measure of neuronal activity is a key technique in neuroscience research.

We’ve developed a number of Molecular Probes ion indicators to track calcium with intense fluorescent signals and a range of wavelength options.

Select calcium indicators for imaging Select calcium indicators for HTS

Ratiometric calcium indicators

Fluorescent Ca²⁺ indicator allows accurate measurement of intracellular calcium concentrations
Ratiometric readout minimizes the effects of photobleaching, leakage, uneven loading, and varying cell thicknesses in mixed populations, delivering more robust and reproducible results

Ratiometric measures have several advantages including reducing the effects of uneven dye loading, leakage of dye and photobleaching.

Additionally, ratiometric calcium indicators reduce the problems associated with measuring Ca²⁺ in cells of unequal thickness. Fura-2 and Indo-1 are typically used to measure changes in calcium concentration by either monitoring excitation or emission, respectively.

Figure 1. Fluorescence excitation spectra of fura-2 (F1200, F6799) in solutions containing 0–39.8 µM free Ca²⁺.

Calcium indicators with resting signal

14-fold signal increase on Ca²⁺ binding
Background fluorescence at resting Ca²⁺ levels

Each of the Oregon Green™ calcium indicators binds intracellular calcium with increasing affinity, providing a sensitivity range to match many applications. Oregon Green probes emit green fluorescence at resting levels of Ca²⁺ and increase their fluorescence intensity 14-fold with increasing Ca²⁺ concentration. They are available in cell-impermeant formulations for loading by microinjection, patch pipette, or pinocytic loading agent, and as dextran conjugates for longer retention in cells.

The cell-permeant formulation can be loaded in cell media and is compatible with microplate assays and flow cytometry as well as imaging assays.

Select Calcium Indicators With Resting Signal

Figure 2. Confocal line scan image of calcium “puffs” in a Xenopus oocyte, using Oregon Green 488 BAPTA-1.

Calcium indicators with no resting signal

100-fold signal increase on Ca²⁺ binding
Minimal fluorescence at resting Ca²⁺ levels

The fluo series of calcium indicators emits minimal fluorescence at resting levels of Ca²⁺, and each increases its fluorescence intensity >100-fold with increasing Ca²⁺ concentration. Each of the fluo dyes binds intracellular calcium with characteristic affinity, providing a sensitivity range to match different Ca²⁺ concentrations. Fluo dyes are available in cell-impermeant formulations for loading by microinjection, patch pipette, or pinocytic loading agent.

Cell-permeant formulations can be loaded in cell media and are compatible with imaging and microplate assays, including HTS.

Select Calcium Indicators With No Resting Signal

Figure 3. HeLa cells loaded with 5 µM Fluo-4.

Long wavelength calcium indicators

Compatible with GFP and green-fluorescent dyes
Rhod-2 localizes to mitochondria

Rhodamine-based calcium indicators comprise a range of probes for large or small changes in Ca²⁺ concentration. They exhibit a 50-fold increase in fluorescence upon calcium binding and offer a range of wavelengths that can be used in conjunction with GFP or green-fluorescent dyes for multiplexing.

Cells can be loaded using membrane-permeant formulations or loading by microinjection, patch pipette, or pinocytic loading agent. Rhod-2 in particular localizes to mitochondria and can be used with imaging, flow cytometry, microplate assays, and HCS platforms.

Select Long Wavelength Calcium Indicators

Figure 4. Live bovine pulmonary artery endothelial cell stained with X-rhod-1 AM.

Calcium indicators selection guides

	Fura-2, AM	Fura-2, pentapotassium	Fura Red, AM	Indo-1, AM	Indo-1, pentapotassium
Readout	Ratiometric excitation wavelength changes in response to calcium binding			Ratiometric emission wavelength changes in response to calcium binding
Preferred method of detection	Excitation wavelength changes			Excitation wavelength changes
Ex/Em (zero calcium) in nM	363/510		436/650	355/475
Ex/Em (high calcium) in nM	363/510		472/650	355/401
Cell permeant or impermeant	Permeant	Impermeant	Permeant	Permeant	Impermeant
Platform	Fluorescence microscopy		Fluorescence microscope, flow cytometry	Fluorescence microscope, flow cytometry
Cat. No.	F1221	F1200	F3021	I1223	I1202

	Oregon Green 488 BAPTA-1, AM	Oregon Green 488 BAPTA-1, Hexapotassium Salt	Oregon Green 488 BAPTA-1 dextran, Potassium Salt, 10,000 MW, Anionic	Oregon Green 488 BAPTA-6F, Hexapotassium Salt, cell impermeant	Oregon Green 488 BAPTA-5N, Hexapotassium Salt, cell impermeant
Readout	14-fold fluorescence intensity increase upon binding Ca²⁺. Visible fluorescence at resting calcium levels makes this dye useful to visualize cell location/structure prior to stimulation.
Range	Detects small changes in intracellular calcium			Detects moderate changes in intracellular calcium	Detects large changes in intracellular calcium
Fluorophore	Oregon Green 488 BAPTA-1			Oregon Green 488 BAPTA-6F	Oregon Green 488 BAPTA-5N
Standard filter set	FITC			FITC
Ex/Em (nm)	494/523			492/517
Bibliography	Citations
Cell permeant or impermeant?	Permeant	Impermeant	Impermeant	Impermeant	Impermeant
Platforms	Flow cytometry, microplate, imaging	Imaging	Imaging	Imaging	Imaging
Usage notes	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent	Dextran reduces cell leakage	Load by microinjection, patch pipette, or pinocytic loading agent
Format	10 x 50 µg	500 µg	5 mg	500 µg	500 µg
Cat. No.	O6807	O6806	O6798	O23990	O6812

	Fluo-4, AM, cell permeant, special packaging	Fluo-4, Pentapotassium Salt, cell impermeant	Fluo-5F, AM, cell permeant, special packaging	Fluo-5F, Pentapotassium Salt, cell impermeant	Fluo-4FF, AM, cell permeant, special packaging	Fluo-4FF, Pentapotassium Salt, cell impermeant
Readout	Large increase (>100-fold) in fluorescence emission intensity upon binding Ca²⁺, minimal fluorescence at resting calcium levels.
Range	Detects small changes in intracellular calcium		Detects moderate changes in intracellular calcium		Detects large changes in intracellular calcium
Fluorophore	Fluo-4		Fluo-5F		Fluo-4FF
Standard filter set	FITC
Ex/Em (nm)	494/506		494/516		494/516
Bibliography	Citations		Citations		Citations
Cell permeant or impermeant?	Permeant	Impermeant	Permeant	Impermeant	Permeant	Impermeant
Platforms	Imaging, flow cytometry, microplate, HCS	Imaging	Imaging, flow cytometry, microplate, HCS	Imaging	Imaging, flow cytometry, microplate, HCS	Imaging
Usage notes	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent
Format	10 x 50 µg	500 µg	10 x 50 µg	500 µg	10 x 50 µg	500 µg
Cat. No.	F14201	F14200	F14222	F14221	F23981	F23980

	Rhod-2, AM, cell permeant	Rhod-2, Tripotassium Salt, cell impermeant	X-Rhod-1, AM, cell permeant	X-Rhod-5F, AM, cell permeant, special packaging	X-Rhod-5F, Tripotassium Salt, cell impermeant
Readout	Large increase in fluorescence emission intensity upon binding Ca²⁺. Can be used in conjunction with GFP or green-fluorescent dyes.
Range	Localizes to mitochondria		Detects small changes in Ca²⁺	Detects large changes in Ca²⁺
Fluorophore	Rhod-2		X-Rhod-1	X-Rhod-5F
Standard filter set	TRITC		Rhodamine	Rhodamine
Ex/Em (nm)	552/581		580/602	581/603
Bibliography	Citations		Citations
Cell permeant or impermeant?	Permeant	Impermeant	Permeant	Permeant	Impermeant
Platforms	Imaging, flow cytometry, microplate, HCS	Imaging	Imaging	Imaging	Imaging
Usage notes	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent	Load in cell media	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent
Format	20 x 50 µg	1 mg	10 x 50 µg	10 x 50 µg	500 µg
Cat. No.	R1244	R14220	X14210	X23985	X23984

Ratiometric Calcium
Indicators

	Fura-2, AM	Fura-2, pentapotassium	Fura Red, AM	Indo-1, AM	Indo-1, pentapotassium
Readout	Ratiometric excitation wavelength changes in response to calcium binding			Ratiometric emission wavelength changes in response to calcium binding
Preferred method of detection	Excitation wavelength changes			Excitation wavelength changes
Ex/Em (zero calcium) in nM	363/510		436/650	355/475
Ex/Em (high calcium) in nM	363/510		472/650	355/401
Cell permeant or impermeant	Permeant	Impermeant	Permeant	Permeant	Impermeant
Platform	Fluorescence microscopy		Fluorescence microscope, flow cytometry	Fluorescence microscope, flow cytometry
Cat. No.	F1221	F1200	F3021	I1223	I1202

Ca²⁺ Indicators With
Resting Signal

	Oregon Green 488 BAPTA-1, AM	Oregon Green 488 BAPTA-1, Hexapotassium Salt	Oregon Green 488 BAPTA-1 dextran, Potassium Salt, 10,000 MW, Anionic	Oregon Green 488 BAPTA-6F, Hexapotassium Salt, cell impermeant	Oregon Green 488 BAPTA-5N, Hexapotassium Salt, cell impermeant
Readout	14-fold fluorescence intensity increase upon binding Ca²⁺. Visible fluorescence at resting calcium levels makes this dye useful to visualize cell location/structure prior to stimulation.
Range	Detects small changes in intracellular calcium			Detects moderate changes in intracellular calcium	Detects large changes in intracellular calcium
Fluorophore	Oregon Green 488 BAPTA-1			Oregon Green 488 BAPTA-6F	Oregon Green 488 BAPTA-5N
Standard filter set	FITC			FITC
Ex/Em (nm)	494/523			492/517
Bibliography	Citations
Cell permeant or impermeant?	Permeant	Impermeant	Impermeant	Impermeant	Impermeant
Platforms	Flow cytometry, microplate, imaging	Imaging	Imaging	Imaging	Imaging
Usage notes	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent	Dextran reduces cell leakage	Load by microinjection, patch pipette, or pinocytic loading agent
Format	10 x 50 µg	500 µg	5 mg	500 µg	500 µg
Cat. No.	O6807	O6806	O6798	O23990	O6812

Ca²⁺ Indicators With No
Resting Signal

	Fluo-4, AM, cell permeant, special packaging	Fluo-4, Pentapotassium Salt, cell impermeant	Fluo-5F, AM, cell permeant, special packaging	Fluo-5F, Pentapotassium Salt, cell impermeant	Fluo-4FF, AM, cell permeant, special packaging	Fluo-4FF, Pentapotassium Salt, cell impermeant
Readout	Large increase (>100-fold) in fluorescence emission intensity upon binding Ca²⁺, minimal fluorescence at resting calcium levels.
Range	Detects small changes in intracellular calcium		Detects moderate changes in intracellular calcium		Detects large changes in intracellular calcium
Fluorophore	Fluo-4		Fluo-5F		Fluo-4FF
Standard filter set	FITC
Ex/Em (nm)	494/506		494/516		494/516
Bibliography	Citations		Citations		Citations
Cell permeant or impermeant?	Permeant	Impermeant	Permeant	Impermeant	Permeant	Impermeant
Platforms	Imaging, flow cytometry, microplate, HCS	Imaging	Imaging, flow cytometry, microplate, HCS	Imaging	Imaging, flow cytometry, microplate, HCS	Imaging
Usage notes	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent
Format	10 x 50 µg	500 µg	10 x 50 µg	500 µg	10 x 50 µg	500 µg
Cat. No.	F14201	F14200	F14222	F14221	F23981	F23980

Long Wavelength Calcium
Indicators

	Rhod-2, AM, cell permeant	Rhod-2, Tripotassium Salt, cell impermeant	X-Rhod-1, AM, cell permeant	X-Rhod-5F, AM, cell permeant, special packaging	X-Rhod-5F, Tripotassium Salt, cell impermeant
Readout	Large increase in fluorescence emission intensity upon binding Ca²⁺. Can be used in conjunction with GFP or green-fluorescent dyes.
Range	Localizes to mitochondria		Detects small changes in Ca²⁺	Detects large changes in Ca²⁺
Fluorophore	Rhod-2		X-Rhod-1	X-Rhod-5F
Standard filter set	TRITC		Rhodamine	Rhodamine
Ex/Em (nm)	552/581		580/602	581/603
Bibliography	Citations		Citations
Cell permeant or impermeant?	Permeant	Impermeant	Permeant	Permeant	Impermeant
Platforms	Imaging, flow cytometry, microplate, HCS	Imaging	Imaging	Imaging	Imaging
Usage notes	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent	Load in cell media	Load in cell media	Load by microinjection, patch pipette, or pinocytic loading agent
Format	20 x 50 µg	1 mg	10 x 50 µg	10 x 50 µg	500 µg
Cat. No.	R1244	R14220	X14210	X23985	X23984

Resources

Molecular Probes Handbook

More information

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