Calbryte™ 630 AM
![Graph illustrates signal-to-noise (SNR) x 100%. ATP dose response was measured in CHO-K1 cells with Calbryte™ 630 AM. CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of 10 µg/ml Calbryte™ 630 AM in HH Buffer with probenecid was added and incubated for 60 min at 37°C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. ATP (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalbryte-630-am%2Fgraph-for-calbryte-630-am_TlRip.webp&w=640&q=75)
![Graph illustrates signal-to-noise (SNR) x 100%. ATP dose response was measured in CHO-K1 cells with Calbryte™ 630 AM. CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of 10 µg/ml Calbryte™ 630 AM in HH Buffer with probenecid was added and incubated for 60 min at 37°C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. ATP (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalbryte-630-am%2Fgraph-for-calbryte-630-am_TlRip.webp&w=640&q=75)
![Graph illustrates signal-to-noise (SNR) x 100%. ATP dose response was measured in CHO-K1 cells with Calbryte™ 630 AM. CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of 10 µg/ml Calbryte™ 630 AM in HH Buffer with probenecid was added and incubated for 60 min at 37°C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. ATP (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalbryte-630-am%2Fgraph-for-calbryte-630-am_TlRip.webp&w=128&q=25)
![The ATP induced intracellular calcium release was measured by Calbryte™ 630 AM. Cells were incubated with Calbryte™ 630 AM dye for 30 min at 37 °C before 10 µM ATP was added into the cells. The baseline was acquired and the rest of the cells were analyzed after the addition of ATP. The response was measured over time. The analysis was done on NovoCyte™ 3000 Flow Cytometer APC Channel.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalbryte-630-am%2Ffigure-for-calbryte-630-am_ibeHC.jpg&w=128&q=25)
![a, b Overexpression of FTO (OE-FTO) in GT1-7 cells as confirmed by qPCR and western blotting (n = 3). c GnRH mRNA levels detected by qPCR in OE-FTO cells (n = 3). d GnRH abundance in OE-FTO cells as determined by ELISA (n = 3). e, f Knockdown of FTO (KD-FTO) in GT1-7 cells as determined by qPCR and western blotting (n = 3). g Expression of GnRH mRNA as determined by qPCR (n = 3). h Protein expression of GnRH in KD-FTO cells as determined by ELISA (n = 3). i Free Ca2+ concentrations of OE-Control and OE-FTO GT1-7 cells as determined by fluorescence of Calbryte-630 with flow cytometry as described in the Methods (n = 3). Left: flow cytometric histograms of Calbryte-630 and gating with no Calbryte-630 probe as background values. Right: quantitative analysis of cell fluorescence for Calbryte-630. j Levels of intracellular free Ca2+ (red colour) between OF-FTO and OE-Control cells as determined by IF (n = 6). The concentration of fluorescence-labelled Calbryte 630 was 5 μM. Scale bars, 100 μm. The bars represent the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, and ns, P > 0.05 versus OE-control or KD-control group by Student’s t test. Source: <b>FTO-mediated m6A demethylation regulates GnRH expression in the hypothalamus via the PLCβ3/Ca2+/CAMK signalling pathway</b> by Zang, S., Yin, X. & Li, P. <em>Commun Biol 6, 1297</em> Dec. 2023.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalbryte-630-am%2Ffigure-for-calbryte-630-am_CFLJu.png&w=96&q=25)
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
Prepare a 2 to 5 mM stock solution of Calbryte™ 630 AM in anhydrous DMSO.
Note: When reconstituted in DMSO, Calbryte™ 630 AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Calbryte™ 630 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Calbryte™ 630 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, Calbryte™ 630 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 Calbryte™ 630 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 Calbryte™ 630 AM working solution into 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 30 to 60 minutes.
Note: Incubating the dye for longer than 1 hour 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 Texas Red filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 600/640 nm cutoff 630 nm.
Common stock solution preparation
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 80.982 µL | 404.911 µL | 809.822 µL | 4.049 mL | 8.098 mL |
5 mM | 16.196 µL | 80.982 µL | 161.964 µL | 809.822 µL | 1.62 mL |
10 mM | 8.098 µL | 40.491 µL | 80.982 µL | 404.911 µL | 809.822 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
/ | = | x | = |
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Calbryte™ 520 AM | 493 | 515 | 0.751 |
Calbryte™ 590 AM | 581 | 593 | - |
Calbryte™-520L AM | 493 | 515 | 0.751 |
Calbryte™-520XL AM | 493 | 515 | 0.751 |
Cal-630™ AM | 609 | 626 | 0.371 |
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