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Multicolor Intracellular Calcium Detection Probes
In this issue
Maleimide Assays
Glycerol Assays
Glycerol 3-Phosphate Assays
Acetylcholinesterase Assays
Nucleic Acid Quantification
Multicolor Intracellular Calcium Detection Probes
Intracellular Nitric Oxide (NO) Assays
Intracellular Total ROS Activity Assays
iFluor™ 700 Dyes
HRP Antibody Labeling Using Buccutite™ Crosslinking Technology
6-JOE SE
AssayWise Letters 2014, Vol. 3(2)
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.
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™ | 492 | 514 | 0.75 | 320 |
| Fluo-3 | 506 | 525 | 0.15 | 390 |
| Fluo-4 | 493 | 515 | 0.16 | 345 |
| Fluo-8® | 490 | 514 | 0.16 | 389 |
- *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 ▲ ▼ |
| 21130 | Cal-520®, AM | 492 | 514 | 10x50 ug |
| 20605 | Cal-520®-Biotin Conjugate | 492 | 514 | 5x50 ug |
| 20606 | Cal-520®-Biocytin Conjugate | 492 | 514 | 5x50 ug |
| 20600 | Cal-520®-Dextran Conjugate *MW 3,000* | 492 | 514 | 1 mg |
| 20601 | Cal-520®-Dextran Conjugate *MW 10,000* | 492 | 514 | 5 mg |
| 20610 | Cal-520® maleimide | 492 | 514 | 100 ug |
| 20609 | Cal-520® NHS Ester | 492 | 514 | 100 ug |
| 21140 | Cal-520®, potassium salt | 492 | 514 | 10x50 ug |
| 21135 | Cal-520®, sodium salt | 492 | 514 | 10x50 ug |
| 21142 | Cal-520FF™, AM | 492 | 514 | 1 mg |
Cal-630™ Calcium Detection Probes
Normalized emission spectra of Cal-520™ (Green), Cal-590™ (Orange) and Cal-630™ (Red).
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.