Cal-520®, AM
Ordering information
| Price | |
| Catalog Number | |
| Availability | In stock |
| Unit Size | |
| Quantity |
Additional ordering information
| Telephone | 1-800-990-8053 |
| Fax | 1-800-609-2943 |
| sales@aatbio.com | |
| International | See distributors |
| Bulk request | Inquire |
| Custom size | Inquire |
| Shipping | Standard overnight for United States, inquire for international |
Physical properties
| Dissociation constant (Kd, nM) | 320 |
| Molecular weight | 1102.95 |
| Solvent | DMSO |
Spectral properties
| Excitation (nm) | 493 |
| Emission (nm) | 515 |
| Quantum yield | 0.751 |
Storage, safety and handling
| H-phrase | H303, H313, H333 |
| Hazard symbol | XN |
| Intended use | Research Use Only (RUO) |
| R-phrase | R20, R21, R22 |
| Storage | Freeze (< -15 °C); Minimize light exposure |
| UNSPSC | 12352200 |
Related products
| Overview |  SDSProtocol |
See also: Calcium Indicators, Multi-Photon Microscopy
Molecular weight 1102.95 | Dissociation constant (Kd, nM) 320 | Excitation (nm) 493 | Emission (nm) 515 | Quantum yield 0.751 |
Cal-520® AM provides a robust homogeneous fluorescence-based assay tool for detecting intracellular calcium mobilization. Cal-520® AM is a new fluorogenic calcium-sensitive dye with a significantly improved signal to noise ratio and intracellular retention compared to the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM). 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 cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Cal-520®. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement, make Cal-520® AM an ideal indicator for the measurement of cellular calcium. The high S/N ratio and better intracellular retention make the Cal-520® calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.
Platform
Fluorescence microscope
| Excitation | FITC |
| Emission | FITC |
| Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
| Excitation | 490 |
| Emission | 525 |
| Cutoff | 515 |
| Recommended plate | Black wall/clear bottom |
| Instrument specification(s) | Bottom read mode/Programmable liquid handling |
Example protocol
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles
Cal-520® AM Stock Solution
Prepare a 2 to 5 mM stock solution of Cal-520® AM in high-quality, anhydrous DMSO.
Note: When reconstituted in DMSO, Cal-520® AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
Cal-520® AM Working Solution
On the day of the experiment, either dissolve Cal-520® AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Cal-520® AM working solution in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Cal-520® AM at a final concentration of 4-5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note: The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Cal-520® AM. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
Note: If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators. A variety of ReadiUse™ Probenecid products, including water-soluble, sodium salt, and stabilized solutions, can be purchased from AAT Bioquest.
SAMPLE EXPERIMENTAL PROTOCOL
Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.
- Prepare cells in growth medium overnight.
On the next day, add 1X Cal-520® AM working solution to your cell plate.
Note: If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.
Incubate the dye-loaded plate in a cell incubator at 37 °C for 1 to 2 hours.
Note: Incubating the dye for longer than 2 hours can improve signal intensities in certain cell lines.
- Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
- Add the stimulant as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 490/525 nm cutoff 515 nm.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Cal-520®, AM to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 90.666 µL | 453.33 µL | 906.659 µL | 4.533 mL | 9.067 mL |
| 5 mM | 18.133 µL | 90.666 µL | 181.332 µL | 906.659 µL | 1.813 mL |
| 10 mM | 9.067 µL | 45.333 µL | 90.666 µL | 453.33 µL | 906.659 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Open in Advanced Spectrum Viewer
Spectral properties
| Excitation (nm) | 493 |
| Emission (nm) | 515 |
| Quantum yield | 0.751 |
Product Family
| Name | Excitation (nm) | Emission (nm) | Quantum yield |
| Cal-520® maleimide | 493 | 515 | 0.751 |
| Cal-520FF™, AM | 493 | 515 | 0.751 |
| Cal-520N™, AM | 493 | 515 | 0.751 |
| Cal-520® amine | 493 | 515 | 0.751 |
| Cal-520® azide | 493 | 515 | 0.751 |
| Cal-520® alkyne | 493 | 515 | 0.751 |
| Cal-590™ AM | 574 | 588 | 0.621 |
| Cal-630™ AM | 609 | 626 | 0.371 |
| Calbryte™ 520 AM | 493 | 515 | 0.751 |
Show More (3) | |||
Images
Figure 1. 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 probenecid (right) under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well plate. 100 µL of 5 µM Fluo-4 AM or Cal 520™ AM in HHBS (with or without probenecid) was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 μL/well) was added using FlexSation to achieve the final indicated concentrations.
Figure 2. Response of endogenous P2Y receptor to ATP in CHO-K cells. CHO-K cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µl of 4 µM Cal 520 ™ AM in HHBS with 1 mM probenecid were added into the wells, and the cells were incubated at 37 °C for 1 hour. The dye loading mediums were replaced with 100 µl HHBS and 1 mM probenecid , then imaged with a fluorescence microscope (Olympus IX71) using FITC channel before and after adding 50 µl of 300 µM ATP .
Figure 3. Two-photon calcium responses to tonal stimuli recorded at 140 ms intervals. Averaged traces (mean and S.E.M.) of ∆F/F0 in 44 neurons stained with Cal-520 AM. The red trace represents responses to 20 kHz stimuli lasting for 7s, and the blue trace shows responses to 20 kHz stimuli lasting for 1s in the same neurons. The off-responses to stimuli lasting for 7 s were significantly larger than the on-responses to stimuli lasting for 1 s (P<0.0001). Source: Auditory cortical field coding long-lasting tonal offsets in mice by Baba et al., Scientific Reports, Sep. 2016.
Figure 4. Functional sperm analysis. (a) Tracks for freely swimming wildtype Prm2+/+ and heterozygous Prm2+/− sperm. (b) Flagellar waveform. Sperm were tethered with their heads to a glass surface and the flagellar waveform was analyzed. One beat cycle was projected. Scale bar: 10 μm. (c) Changes in the intracellular Ca2+ concentration in Prm2+/+, Prm2+/−, and Prm2−/− sperm. Sperm have been loaded with Cal520-AM and stimulated with K8.6 (blue), 10 mM 8-Br-cAMP (red), 10 mM NH4Cl (green), or 2 μM ionomycin (light blue). Experiments have been measured using the stopped-flow technique. (d) Loading of sperm with Cal520-AM. Loading of Prm2+/−, and Prm2−/− sperm was tested using fluorescence microscopy. Scale bar = 20 μm. Source: Re-visiting the Protamine-2 locus: deletion, but not haploinsufficiency, renders male mice infertile by Schneider et al., Scientific Reports, Nov. 2016.
Figure 5. Selectivity of V1 neurons. A) Neurons stained with Cal-520 but not with SR-101 in the V1 of a wild-type mouse (left) and a Pcdhα1,12 mouse (right). The image was obtained using a two-photon microscope. B) Sample traces of neuronal calcium responses to moving grating patterns in eight directions (from -45° to 270° in 45° steps) for 2 s in a wild-type mouse (left) and a Pcdh-α1,12 mouse (right). C) Cumulative distributions of the orientation selectivity index (OSI, left) and direction selectivity index (DSI, right) of neurons obtained from three wild-type mice and three Pcdh-α1,12 mice. The OSI was obtained from1698 and 1342 neurons, respectively. The DSI was obtained from 365 and 302 neurons with an OSI > 0.45, respectively. There was no significant difference in the cumulative distribution of the OSI or DSI between wild-type and Pcdhα1,12 mice. Source: Molecular diversity of clustered protocadherin-α required for sensory integration and short-term memory in mice by Yamagishi et al., Scientific Reports, June 2018.
![Loading induced chromatin condensation is regulated by calcium signaling. (A) Representative [Ca<sup>2+</sup>]<sub>i</sub> oscillations (red arrows) in MSCs as a function of time (bar = 100 μm). Addition of ATP (B) or application of 30s DL (C) decreased the time between peeks and increased number of peaks observed in 10 min (n = ~15, *p < 0.05 vs. CM control (0% strain/0mM ATP, mean ± s.d.). (D,E) Pretreatment with BAPTA or KN62 blocked load-induced chromatin condensation, whereas pretreatment with thapsigargin (TG, F) had no effect and Verapamil (VP, G) blocked only the short term increase in CCP (at 600s of DL) (red line: CM control, green line: 600s DL, blue line: 3 h DL, n = ~20 per condition, *p < 0.05 vs. CM control, mean ± s.e.m.). (H) Schematic illustration outlining the operative signaling pathways controlling chromatin condensation with short term (600s) or long term (3h) loading (⊗ a component that is not on the critical path for load induced chromatin condensation at that time point. ATP: Adenosine triphosphate, HMCLs: hemichannels, P2YR: P2Y purinergic receptors, P2XR: P2X purinergic receptors, ER: endoplasmic reticulum, Ca: calcium. CBPs: calcium binding proteins, VGCC: voltage-gated calcium channels, TRPV4: Transient receptor potential cation channel subfamily V member 4, PIEZO: Piezo-type mechanosensitive ion channels, HDAC: Histone deacetylase, MTF: Histone methyltransferase, LICC: load induced chromatin condensation). Source: <strong>Biophysical Regulation of Chromatin Architecture Instills a Mechanical Memory in Mesenchymal Stem Cells</strong> by Heo et al., <em>Scientific Reports</em>, Nov. 2015.](https://images.aatbio.com/products/figures-and-data/cal-520-am/figure-for-cal-520-am_dwIXN.jpg)
Figure 6. Loading induced chromatin condensation is regulated by calcium signaling. (A) Representative [Ca2+]i oscillations (red arrows) in MSCs as a function of time (bar = 100 μm). Addition of ATP (B) or application of 30s DL (C) decreased the time between peeks and increased number of peaks observed in 10 min (n = ~15, *p < 0.05 vs. CM control (0% strain/0mM ATP, mean ± s.d.). (D,E) Pretreatment with BAPTA or KN62 blocked load-induced chromatin condensation, whereas pretreatment with thapsigargin (TG, F) had no effect and Verapamil (VP, G) blocked only the short term increase in CCP (at 600s of DL) (red line: CM control, green line: 600s DL, blue line: 3 h DL, n = ~20 per condition, *p < 0.05 vs. CM control, mean ± s.e.m.). (H) Schematic illustration outlining the operative signaling pathways controlling chromatin condensation with short term (600s) or long term (3h) loading (⊗ a component that is not on the critical path for load induced chromatin condensation at that time point. ATP: Adenosine triphosphate, HMCLs: hemichannels, P2YR: P2Y purinergic receptors, P2XR: P2X purinergic receptors, ER: endoplasmic reticulum, Ca: calcium. CBPs: calcium binding proteins, VGCC: voltage-gated calcium channels, TRPV4: Transient receptor potential cation channel subfamily V member 4, PIEZO: Piezo-type mechanosensitive ion channels, HDAC: Histone deacetylase, MTF: Histone methyltransferase, LICC: load induced chromatin condensation). Source: Biophysical Regulation of Chromatin Architecture Instills a Mechanical Memory in Mesenchymal Stem Cells by Heo et al., Scientific Reports, Nov. 2015.
![CaV1.3-mediated Ca2+ signalling emerges in LNCaP during CSFBS culturing. (A) Pseudocolour micrographs of Cal-520 AM-loaded LNCaP (cultured in media containing normal FBS) during live-cell recordings. Pseudocolour scale indicating increased fluorescence intensity from blue–green–yellow–red, indicating increased intracellular [Ca2+]. Fluorescence micrographs in normal Hanks' (0 s) and during perfusion with high K+ Hanks containing Bay K 8644 (10 µM, denoted by solid horizontal bar) (100 s) are shown. Five cells (1–5, white arrows) were selected and ΔF/F0 measured then plotted on a fluorescence intensity-time graph (coloured lines, lower panel) (N = 5 recordings). (B) Pseudocolour micrographs showing CSFBS-cultured LNCaP (10 days) pre- (0 s) and during exposure (100 s) to high K+/Bay K 8644. Fluorescence intensity of 5 selected cells demonstrating Ca2+-transients in 4 of the 5 cells (lower panel) (N = 3 recordings). (C) Mean percentage of responding cells per recording is shown at several time points during CSFBS along with matched time controls. Data analysed with 1-way ANOVA with Šídák's multiple comparison test, p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) (N = 3). (D) Nifedipine (1 µM) significantly reduced the percentage of high K+/Bay K 8644-responding cells after 10 day CSFBS. Data was analysed with unpaired t-test where p < 0.001 (***) (N = 5 recordings). (E) Significant knockdown of CACNA1D by siRNA transfection was achieved in CSBFS-treated LNCaP (14 days, Mann–Whitney, N = 4, p < 0.05 (*). (F) The percentage of high K+/Bay K8644-responsive LNCaP in siRNA-LNCaP was markedly reduced compared with scrambled controls, (p = 0.0578, unpaired t-test, N = 3). Source: <b>CACNA1D overexpression and voltage-gated calcium channels in prostate cancer during androgen deprivation</b> by McKerr, Niamh et.al., <em>Scientific Reports</em>, March 2023](https://images.aatbio.com/products/figures-and-data/cal-520-am/figure-for-cal-520-am_Tjr1o.png)
Figure 7. CaV1.3-mediated Ca2+ signalling emerges in LNCaP during CSFBS culturing. (A) Pseudocolour micrographs of Cal-520 AM-loaded LNCaP (cultured in media containing normal FBS) during live-cell recordings. Pseudocolour scale indicating increased fluorescence intensity from blue–green–yellow–red, indicating increased intracellular [Ca2+]. Fluorescence micrographs in normal Hanks' (0 s) and during perfusion with high K+ Hanks containing Bay K 8644 (10 µM, denoted by solid horizontal bar) (100 s) are shown. Five cells (1–5, white arrows) were selected and ΔF/F0 measured then plotted on a fluorescence intensity-time graph (coloured lines, lower panel) (N = 5 recordings). (B) Pseudocolour micrographs showing CSFBS-cultured LNCaP (10 days) pre- (0 s) and during exposure (100 s) to high K+/Bay K 8644. Fluorescence intensity of 5 selected cells demonstrating Ca2+-transients in 4 of the 5 cells (lower panel) (N = 3 recordings). (C) Mean percentage of responding cells per recording is shown at several time points during CSFBS along with matched time controls. Data analysed with 1-way ANOVA with Šídák's multiple comparison test, p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) (N = 3). (D) Nifedipine (1 µM) significantly reduced the percentage of high K+/Bay K 8644-responding cells after 10 day CSFBS. Data was analysed with unpaired t-test where p < 0.001 (***) (N = 5 recordings). (E) Significant knockdown of CACNA1D by siRNA transfection was achieved in CSBFS-treated LNCaP (14 days, Mann–Whitney, N = 4, p < 0.05 (*). (F) The percentage of high K+/Bay K8644-responsive LNCaP in siRNA-LNCaP was markedly reduced compared with scrambled controls, (p = 0.0578, unpaired t-test, N = 3). Source: CACNA1D overexpression and voltage-gated calcium channels in prostate cancer during androgen deprivation by McKerr, Niamh et.al., Scientific Reports, March 2023
Figure 8. Spectral image data from three single-label control samples were analyzed to construct a spectral library. (A) Representation of HSI image data from HASMCs labeled with the nuclear label, NucBlue. For visualization purposes, all wavelength bands have been summed to represent a total or summed fluorescence intensity image, and the intensity range linearly adjusted from 0 to 6300 A.U. for display. (B) A summed fluorescence intensity representation of HSI image data from a separate sample of unlabeled HASMCs with intensity range linearly adjusted from 0 to 315 A.U. for display. (C) A summed fluorescence intensity representation of HSI image data from a separate sample of HASMCs labeled with Cal 520 with intensity range linearly adjusted from 0 to 550 A.U. (D) A spectral library was generated by selecting a region of high signal intensity within each of the single-label control spectral images (A–C), extracting the pixel-averaged spectrum, and normalizing to a peak value of unity. (E) Comparison of measured spectra for NucBlue (blue squares) and Cal 520 (green triangles) to reported spectra. The Cal 520 spectrum was supplied from AAT Bioquest, while the NucBlue spectrum was approximated as that of DAPI and obtained using the Semrock Searchlight spectral plotting tool. Source: Comparing Performance of Spectral Image Analysis Approaches for Detection of Cellular Signals in Time-Lapse Hyperspectral Imaging Fluorescence Excitation-Scanning Microscopy by Parker et al., Bioengineering, April 2023.
Citations
View all 489 citations: Citation Explorer
Regulation of specific abnormal calcium signals in the hippocampal CA1 and primary cortex M1 alleviates the progression of temporal lobe epilepsy
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Analysis of Fatty Acid Metabolism in Fetal and Failing Hearts by Single-Cell RNA Sequencing Revealed SLC27A6 as a Critical Gene in Heart Maturation
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Journal: Acta Cardiologica Sinica (2023): 580
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Electroconductive and mechano-competent PUCL@ CNT nanohybrid scaffolds guiding neuronal specification of neural stem/progenitor cells
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Journal: Chemical Engineering Journal (2023): 143125
Comparing Performance of Spectral Image Analysis Approaches for Detection of Cellular Signals in Time-Lapse Hyperspectral Imaging Fluorescence Excitation-Scanning Microscopy
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Journal: Bioengineering (2023): 642
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Journal: The Journal of Clinical Endocrinology \& Metabolism (2023): dgad525
The Effects of Mechanical Load on Chondrogenic Responses of Bone Marrow Mesenchymal Stem Cells and Chondrocytes Encapsulated in Chondroitin Sulfate-Based Hydrogel
Authors: Uzieliene, Ilona and Bironaite, Daiva and Bagdonas, Edvardas and Pachaleva, Jolita and Sobolev, Arkadij and Tsai, Wei-Bor and Kvederas, Giedrius and Bernotiene, Eiva
Journal: International Journal of Molecular Sciences (2023): 2915
Authors: Uzieliene, Ilona and Bironaite, Daiva and Bagdonas, Edvardas and Pachaleva, Jolita and Sobolev, Arkadij and Tsai, Wei-Bor and Kvederas, Giedrius and Bernotiene, Eiva
Journal: International Journal of Molecular Sciences (2023): 2915
High-Throughput Screening Assay for Detecting Drug-Induced Changes in Synchronized Neuronal Oscillations and Potential Seizure Risk Based on Ca2+ Fluorescence Measurements in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neuronal 2D and 3D Cultures
Authors: Lu, Hua-Rong and Seo, Manabu and Kreir, Mohamed and Tanaka, Tetsuya and Yamoto, Rie and Altrocchi, Cristina and van Ammel, Karel and Tekle, Fetene and Pham, Ly and Yao, Xiang and others,
Journal: Cells (2023): 958
Authors: Lu, Hua-Rong and Seo, Manabu and Kreir, Mohamed and Tanaka, Tetsuya and Yamoto, Rie and Altrocchi, Cristina and van Ammel, Karel and Tekle, Fetene and Pham, Ly and Yao, Xiang and others,
Journal: Cells (2023): 958
Functional organization of visual responses in the octopus optic lobe
Authors: Pungor, Judit R and Allen, V Angelique and Songco-Casey, Jeremea O and Niell, Cristopher M
Journal: bioRxiv (2023): 2023--02
Authors: Pungor, Judit R and Allen, V Angelique and Songco-Casey, Jeremea O and Niell, Cristopher M
Journal: bioRxiv (2023): 2023--02
Reduction in Junctophilin 2 Expression in Cardiac Nodal Tissue Results in Intracellular Calcium-Driven Increase in Nodal Cell Automaticity
Authors: Landstrom, Andrew P and Yang, Qixin and Sun, Bo and Perelli, Robin M and Bidzimou, Minu-Tshyeto and Zhang, Zhushan and Aguilar-Sanchez, Yuriana and Alsina, Katherina M and Cao, Shuyi and Reynolds, Julia O and others,
Journal: Circulation: Arrhythmia and Electrophysiology (2023): e010858
Authors: Landstrom, Andrew P and Yang, Qixin and Sun, Bo and Perelli, Robin M and Bidzimou, Minu-Tshyeto and Zhang, Zhushan and Aguilar-Sanchez, Yuriana and Alsina, Katherina M and Cao, Shuyi and Reynolds, Julia O and others,
Journal: Circulation: Arrhythmia and Electrophysiology (2023): e010858
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Application notes
A Meta-Analysis of Common Calcium Indicators
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Cal-520 ® , Cal-590 ™, and Cal-630™ Calcium Detection Reagents
Calibration Protocol for Fluorescent Calcium Indicators
Novel improved Ca2+ indicator dyes on the market-a comparative study of novel Ca2+ indicators with fluo-4
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Cal-520 ® , Cal-590 ™, and Cal-630™ Calcium Detection Reagents
Calibration Protocol for Fluorescent Calcium Indicators
Novel improved Ca2+ indicator dyes on the market-a comparative study of novel Ca2+ indicators with fluo-4