The study of the function of Ca2+ ions inside cells is one the most dynamic areas of modern cell biology. Ca2+ is known to control neurosynaptic transmission, secretion of hormones, muscle contraction, and a myriad of physiological functions. It is suspected to be involved in cell division, movements of non-muscle cells, as well as memory and learned patterns of the nervous system. In all cases, localized fluctuations in cytosolic free Ca2+ levels inside cells are believed to control these functions. Techniques for the measurement and manipulation of Ca2+ are therefore crucial and have been advancing rapidly largely as a result of the invention of fluorescent ion indicators. We list our fluorescent calcium indicators by grouping them according to the color of the light they emit on binding calcium, namely Red, Green, and Near UV. We also sell non-fluorescent BAPTA derivatives. Standard instrumentation is readily available in most laboratories and core imaging facilities for compatibility with the multitude of green fluorescent probes in the market. Photochemical constraints and low quantum yields have contributed to the relatively small amounts of red-emitting probes compared to the green-emitting probes.
Our new red calcium indicator, Ion Red Calcium 660 (IRC-660) is under development. Upon completion, this product will have properties that exceed the normal criteria for fluorescent calcium indicators:
*The AM is for non-invasive loading and generates the salt inside the cell by non-specific esterases.
Protocol- to be written by Dr. Joe Kao (U. Maryland).
Modifications of IRC-660 can be made for other applications upon request in a CUSTOM SYNTHESIS.
- Good loading
- Good quantum yield
- Great pKa
- Good extinction coefficient (22,000)
Advantages over green emitting fluorescent indicators1The Red-emitting wavelength region is attractive for biological research because it affords:
- Higher tissue penetration due to reduced scattering and reduced absorption by endogenous fluorophores (usually green)
- Lower phototoxicity
- Reduced spectral overlap by spectrally broad autofluorescence
- Work in an optical window where water and hemoglobin absorption is reduced
- Monitoring Ca2+ in cells or tissues expressing yellow or green fluorescent protein or labelled with other green-emitting fluorophores
DataTable 4.1 Ion Indicators Calcium Indicators
|ION Calcium Red 660 K+ Salt||1056||580 nm||660 nm||40||0.400||H2O||5.7|
|ION Calcium Red 660 AM||1190||*||*||*||*||DMSO||*|
FLUO SERIESMost useful and inexpensive lasers operate in the green part of the visible spectrum and that has made green-emitting fluorescent calcium indicators very useful for confocal microscopy, flow cytometry, and screening. The popular green-emitting fluorescent calcium indicators for screening are FLUO-2, FLUO-3 and FLUO-4. They are all derived from the original FLUO compound (Figure 1) invented by Minta and Tsien (1989).2
|FLUO-2||x = H||R = CH3|
|FLUO-3||x = Cl||R = CH3|
|FLUO-4||x = F||R = CH3|
|Indicator||MW||Excitation (nm)||Emission (nm)||Kd||Extinction||Solubility|
|FLUO-2 K+ Salt||891||490||515||230||80,000||H2O|
|FLUO-3 K+ Salt||960||506||526||390||80,000||H2O|
|FLUO-4 K+ Salt||927||490||515||345||80,000||H2O|