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.

Calcium Imaging Kits

  • Optimized for fluorescence imaging experiments
  • Key reagents and protocols provided
  • Choice of red- or green-fluorescent reporters

To provide an easy introduction to imaging Ca2+ flux using either green- or red-fluorescent dyes, we’ve developed both rhodamine-based and fluo-4–based calcium imaging kits.

Both dyes are optimized for imaging small changes in Ca2+ concentration, and they exhibit a 50-fold (for the green dye) or 100-fold (for the red dye) increase in fluorescence upon calcium binding. The choice of two different wavelengths provides options for multiplexing.

While cells are in culture medium, dye may be loaded using PowerLoad™ reagent, and the kits provide sufficient reagents for approximately 50 coverslips using standard conditions.

 

Figure 1. Differentiated NG108-15 cells grown in culture and loaded with fluo-4 acetoxymethyl (AM) ester to show intracellular calcium concentration.

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.

  Oregon Green® 488 BAPTA-1

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 BPAE 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

 
Readout >100-fold increase in fluorescence emission intensity upon binding Ca2+, minimal fluorescence at resting calcium levels >50-fold increase in fluorescence emission intensity upon binding Ca2+; can be used in conjunction with GFP or green-fluorescent dyes
Range Detects small changes in intracellular calcium
Fluorophore Fluo-4 Rhod-3
Standard filter set FITC TRITC
Ex/Em (nm) 494/506 556/579
Cell permeant or impermeant? Permeant
Platforms Fluorescence microscopy
Usage notes Cells are loaded in medium using PowerLoad™ reagent and probenecid (supplied in the kit)
Format 1 kit/50 coverslips 1 kit/50 coverslips
Cat. No. F10489 R10145
 
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