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.

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.

 Fluorescence excitation spectra of fura-2
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.

Confocal line scan image of calcium “puffs”

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.

HeLa cells loaded with 5 µM Fluo-4

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.

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

Calcium indicators selection guides

 Fura-2, AMFura-2, pentapotassiumFura Red, AMIndo-1, AMIndo-1, pentapotassium
ReadoutRatiometric excitation wavelength changes in response to calcium bindingRatiometric emission wavelength changes in response to calcium binding
Preferred method of detectionExcitation wavelength changesExcitation wavelength changes
Ex/Em (zero calcium) in nM363/510436/650355/475
Ex/Em (high calcium) in nM363/510472/650355/401
Cell permeant or impermeantPermeantImpermeantPermeantPermeantImpermeant
PlatformFluorescence microscopyFluorescence microscope, flow cytometryFluorescence microscope, flow cytometry
Cat. No.F1221F1200F3021I1223I1202
 
Readout14-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.
RangeDetects small changes in intracellular calciumDetects moderate changes in intracellular calciumDetects large changes in intracellular calcium
FluorophoreOregon Green 488 BAPTA-1Oregon Green 488 BAPTA-6FOregon Green 488 BAPTA-5N
Standard filter setFITCFITC
Ex/Em (nm)494/523492/517
BibliographyCitations
Cell permeant or impermeant?PermeantImpermeantImpermeantImpermeantImpermeant
PlatformsFlow cytometry, microplate, imagingImagingImagingImagingImaging
Usage notesLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agentDextran reduces cell leakageLoad by microinjection, patch pipette, or pinocytic loading agent
Format10 x 50 µg500 µg5 mg500 µg500 µg
Cat. No. O6807O6806O6798O23990O6812
 
ReadoutLarge increase (>100-fold) in fluorescence emission intensity upon binding Ca2+, minimal fluorescence at resting calcium levels.
RangeDetects small changes in intracellular calciumDetects moderate changes in intracellular calciumDetects large changes in intracellular calcium
FluorophoreFluo-4Fluo-5FFluo-4FF
Standard filter setFITC
Ex/Em (nm)494/506494/516494/516
BibliographyCitationsCitationsCitations
Cell permeant or impermeant?PermeantImpermeantPermeantImpermeantPermeantImpermeant
PlatformsImaging, flow cytometry, microplate, HCSImagingImaging, flow cytometry, microplate, HCSImagingImaging, flow cytometry, microplate, HCSImaging
Usage notesLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agentLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agentLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agent
Format10 x 50 µg500 µg10 x 50 µg500 µg10 x 50 µg500 µg
Cat. No.F14201F14200F14222F14221F23981F23980
 X-Rhod-1, AM, cell permeant
ReadoutLarge increase in fluorescence emission intensity upon binding Ca2+. Can be used in conjunction with GFP or green-fluorescent dyes.
RangeLocalizes to mitochondriaDetects small changes in Ca2+Detects large changes in Ca2+
FluorophoreRhod-2X-Rhod-1X-Rhod-5F
Standard filter setTRITCRhodamineRhodamine
Ex/Em (nm)552/581580/602581/603
BibliographyCitationsCitations
Cell permeant or impermeant?PermeantImpermeantPermeantPermeantImpermeant
PlatformsImaging, flow cytometry, microplate, HCSImagingImagingImagingImaging
Usage notesLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agentLoad in cell mediaLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agent
Format20 x 50 µg1 mg10 x 50 µg10 x 50 µg500 µg
Cat. No. R1244R14220X14210X23985X23984
 Fura-2, AMFura-2, pentapotassiumFura Red, AMIndo-1, AMIndo-1, pentapotassium
ReadoutRatiometric excitation wavelength changes in response to calcium bindingRatiometric emission wavelength changes in response to calcium binding
Preferred method of detectionExcitation wavelength changesExcitation wavelength changes
Ex/Em (zero calcium) in nM363/510436/650355/475
Ex/Em (high calcium) in nM363/510472/650355/401
Cell permeant or impermeantPermeantImpermeantPermeantPermeantImpermeant
PlatformFluorescence microscopyFluorescence microscope, flow cytometryFluorescence microscope, flow cytometry
Cat. No.F1221F1200F3021I1223I1202
 
Readout14-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.
RangeDetects small changes in intracellular calciumDetects moderate changes in intracellular calciumDetects large changes in intracellular calcium
FluorophoreOregon Green 488 BAPTA-1Oregon Green 488 BAPTA-6FOregon Green 488 BAPTA-5N
Standard filter setFITCFITC
Ex/Em (nm)494/523492/517
BibliographyCitations
Cell permeant or impermeant?PermeantImpermeantImpermeantImpermeantImpermeant
PlatformsFlow cytometry, microplate, imagingImagingImagingImagingImaging
Usage notesLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agentDextran reduces cell leakageLoad by microinjection, patch pipette, or pinocytic loading agent
Format10 x 50 µg500 µg5 mg500 µg500 µg
Cat. No. O6807O6806O6798O23990O6812
 
ReadoutLarge increase (>100-fold) in fluorescence emission intensity upon binding Ca2+, minimal fluorescence at resting calcium levels.
RangeDetects small changes in intracellular calciumDetects moderate changes in intracellular calciumDetects large changes in intracellular calcium
FluorophoreFluo-4Fluo-5FFluo-4FF
Standard filter setFITC
Ex/Em (nm)494/506494/516494/516
BibliographyCitationsCitationsCitations
Cell permeant or impermeant?PermeantImpermeantPermeantImpermeantPermeantImpermeant
PlatformsImaging, flow cytometry, microplate, HCSImagingImaging, flow cytometry, microplate, HCSImagingImaging, flow cytometry, microplate, HCSImaging
Usage notesLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agentLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agentLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agent
Format10 x 50 µg500 µg10 x 50 µg500 µg10 x 50 µg500 µg
Cat. No.F14201F14200F14222F14221F23981F23980
 X-Rhod-1, AM, cell permeant
ReadoutLarge increase in fluorescence emission intensity upon binding Ca2+. Can be used in conjunction with GFP or green-fluorescent dyes.
RangeLocalizes to mitochondriaDetects small changes in Ca2+Detects large changes in Ca2+
FluorophoreRhod-2X-Rhod-1X-Rhod-5F
Standard filter setTRITCRhodamineRhodamine
Ex/Em (nm)552/581580/602581/603
BibliographyCitationsCitations
Cell permeant or impermeant?PermeantImpermeantPermeantPermeantImpermeant
PlatformsImaging, flow cytometry, microplate, HCSImagingImagingImagingImaging
Usage notesLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agentLoad in cell mediaLoad in cell mediaLoad by microinjection, patch pipette, or pinocytic loading agent
Format20 x 50 µg1 mg10 x 50 µg10 x 50 µg500 µg
Cat. No. R1244R14220X14210X23985X23984