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.

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.

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.

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
 
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 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. R1245MP R1244 X14210 X23985 X23984