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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 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. |
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.
Figure 2. Confocal line scan image of calcium “puffs” in a Xenopus oocyte, using Oregon Green 488 BAPTA-1.
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.
Figure 3. HeLa cells loaded with 5 µM Fluo-4.
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.
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 |
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 |
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 |
X-Rhod-1, AM, cell permeant | |||||
---|---|---|---|---|---|
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 |
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 |
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 |
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 |
X-Rhod-1, AM, cell permeant | |||||
---|---|---|---|---|---|
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 |
For Research Use Only. Not for use in diagnostic procedures.
For Research Use Only. Not for use in diagnostic procedures.