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Multicolor Intracellular Calcium Detection Probes

Cal-520™ Calcium Detection Probes


Cal-520™ AM is a new fluorogenic calcium-sensitive dye with a significantly improved signal to background ratio and intracellular retention compared to the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM). Cal-520™ AM provides the most robust homogeneous fluorescence-based assay tool for detecting intracellular calcium mobilization. Cells expressing a GPCR or calcium channel of interest that signals through calcium can be preloaded with Cal-520™ AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Cal-520™ AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increases the fluorescence of Cal-520™ AM. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement, make Cal-520™ AM an ideal indicator for the measurement of intracellular calcium. The high signal to background ratio and better intracellular retention make the Cal-520™ AM calcium assay an ideal tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.


Calcium response comparison of Cal-520™-dextran with Calcium Green™ -1-dextran in the presence of saturated calcium citrate.
AAT Bioquest offers a full range of Cal-520™ calcium detection probes. Cal-520FF™ is used for monitoring calcium in high concentration. The dextran forms of our calcium indicators show a dramatic reduction in both leakage and compartmentalization compared to the AM ester forms. Among the fluorescent calcium indicator dextran conjugates, Cal-520™-dextran conjugates might be the best choice due to their high fluorescence quantum yields and large fluorescence enhancements by calcium. Compared to Oregon Green® BAPTA-1 dextran, Cal-520™-dextran exhibits much lower background and much larger calcium-induced fluorescence enhancement. Cal-520™-biotin and Cal-520™-biocytin conjugates have great calcium responses. Their avidin and streptavidin complexes have sensitive calcium responses as well. Conceivably Cal-520™-biotin and Cal-520™-biocytin conjugates could be bound to an IgG-avidin or an IgG-streptavidin conjugate for monitoring calcium change spatially for a specific target. Our amine-reactive Cal-520™ NHS ester and thiol-reactive Cal-520™ maleimide could be coupled to an IgG or other carrier molecules to prepare a calcium-sensitive bioconjugates for monitoring calcium change spatially for a specific target.


Responses of endogenous P2Y receptor to ATP in CHO-M1 cells without probenecid. CHO-M1 cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar 96-well black wall/clear bottom plate. 100 µL of 4 µM Fluo-4 AM (left), Cal-520™ AM (right) in HHBS was added into the wells, and the cells were incubated at 37 °C for 2 hours. The dye loading medium was replaced with 100 µL HHBS and 50 µL of 300 µM ATP were added. The cells were imaged with a fluorescence microscope (Olympus IX71) using FITC channel.



ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-520™ AM (red curve), or Fluo-4 AM (blue curve) respectively with (left) or without proben-ecid (right) under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a Costar 96-well black wall/clear bottom plate. 100 µL of 5 µM Fluo-4 AM or Cal 520™ AM in HHBS (with or without 2.5 mm probenecid) was added into the cells, and the cells were incubated at 37 °C for 2 hours. ATP (50 µL/well) was added using FlexStation® to achieve the final indicated concentra-tions.

 

Table 1. Spectral Comparison of Fluo-3, Fluo-4, Fluo-8® and Cal-520

Dye
Ex (nm)
Em (nm)
QY*
Kd (nM)
Cal-520™ 4925140.75320
Fluo-3 5065250.15390
Fluo-4 4935150.16345
Fluo-8®4905140.16389
  1. *QY = Fluorescence Quantum Yield in the presence of 5 mM calcium citrate.

 

Cal-590™ Calcium Detection Probes


Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding calcium have enabled researchers to investigate changes in intracellular free calcium concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Rhod-2 AM is most commonly used among the red fluorescent calcium indicators. However, Rhod-2 AM is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses.


Responses of endogenous P2Y receptor to ATP in CHO-K1 cells. CHO-K1 cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar 96-well black wall/clear bottom plate. 100 µL of 4 µM Cal-590™ AM (Cat# 20510) in HHBS with 1 mM probenecid were added into the wells, and the cells were incubated at 37 °C for 2 hours. The dye loading mediums were replaced with 100 µL HHBS and 1 mM probenecid , then imaged with a fluorescence microscope (Olympus IX71) using TRITC channel before and after adding 50 µL of 300 µM ATP .


Cal-590™ has been developed to improve Rhod-2 AM cell loading and calcium response while maintaining the similar spectral wavelengths of Rhod-2 AM, making it compatible with TRITC/Cy3® filter set. In CHO and HEK cells, the cellular calcium response of Cal-590™ is much more sensitive than that of Rhod-2 AM. The spectra of Cal-590™ is well separated from those of FITC, Alexa Fluor® 488 and GFP, making it an ideal calcium probe for multiplexing intracellular assays with GFP cell lines or FITC/Alexa Fluor® 488 labeled antibodies.


Left: Fluorescence emission spectra of Cal-590™ in solutions containing 0 to 39 µM free Ca2+. Right: ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-590™ AM (Red) and Rhod-2, AM (Blue) under the same condi-tions. CHO-K1 cells were seeded overnight at the cell density of 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom plate. 100 µL of 5 µg/mL Cal-590™ AM or Rhod-2 AM with 2.5 mM probenecid was added into the cells, and the cells were incubated at 37 oC for 1 hour. ATP (50 µL/well) was added by FlexStation® (Molecular Devices) to achieve the final indicated concentrations.

 

Table 2. Cal-520™ and Cal-590™ Calcium Detection Probes

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
21130Cal-520®, AM49251410x50 ug
20605Cal-520®-Biotin Conjugate4925145x50 ug
20606Cal-520®-Biocytin Conjugate4925145x50 ug
20600Cal-520®-Dextran Conjugate *MW 3,000*4925141 mg
20601Cal-520®-Dextran Conjugate *MW 10,000*4925145 mg
20610Cal-520® maleimide492514100 ug
20609Cal-520® NHS Ester492514100 ug
21140Cal-520®, potassium salt49251410x50 ug
21135Cal-520®, sodium salt49251410x50 ug
21142Cal-520FF™, AM4925141 mg

 

Cal-630™ Calcium Detection Probes



Normalized emission spectra of Cal-520™ (Green), Cal-590™ (Orange) and Cal-630™ (Red).
X-Rhod-1 is commonly used as a red fluorescent calcium indicator. However, X-Rhod-1 is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses. In addition, X-Rhod-1 is mostly localized in mitochondria, thus giving low signal/background ratio. Cal-630™ has been developed to improve X-Rhod-1 cell loading and calcium response while maintaining the similar spectral wavelengths of X-Rhod-1, making it compatible with Texas Red® filter set. In CHO and HEK cells, the cellular calcium response of Cal-630™ is much more sensitive than that of X-Rhod-1. The maximum emission wavelength of Cal-630™ is well separated from those of FITC, Alexa Fluor® 488 and GFP, mak-ing it an ideal calcium probe for multiplexing intracellular assays with GFP cell lines or FITC/Alexa Fluor® 488 labeled antibodies.




Left: Fluorescence emission spectra of Cal-630™ in solutions containing 0 to 39 µM free Ca2+. Right: ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells measured with Cal-630™ AM (Cat# 20530). CHO-K1 cells were seeded overnight at the cell density of 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom plate. 100 µL of 10 µg/mL Cal-630™ AM with 2.0 mM probenecid was added into the cells, and the cells were incubated at 37 °C for 2 hours. ATP (50 µL/well) was added by FlexStation® (Molecular Devices) to achieve the final indicated concentrations.



Responses of endogenous P2Y receptor to ATP in CHO-K1 cells. CHO-K1 cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom plate. 100 µL of 4 µM Cal-630™ AM (Cat# 20530) in HHBS with 1 mM probenecid were added into the wells, and the cells were incubated at 37 °C for 2 hours. The dye loading mediums were replaced with 100 µL HHBS and 1 mM probenecid , then imaged with a fluorescence microscope (Olympus IX71) using TRITC channel before and after adding 50 µL of 300 µM ATP.

 

Table 3. Cal-630™ Calcium Detection Probes

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
20530Cal-630™ AM6086265x50 ug
20531Cal-630™ AM60862610x50 ug
20538Cal-630™, potassium salt6086265x50 ug
20535Cal-630™, sodium salt6086265x50 ug