Calbryte™ 630 AM
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
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.
Calculators
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 | = |
Spectrum
Product family
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 |
Citations
Authors: Li, Jizhen and Zhao, Lingling and Wu, Zerui and Huang, Shirui and Wang, Junyu and Chang, Yuanyuan and Liu, Li and Jin, Honglei and Lu, Jianglong and Huang, Chuanshu and others,
Journal: Journal of Experimental \& Clinical Cancer Research (2024): 231
Authors: Acreman, Samuel and Ma, Jinfang and Denwood, Geoffrey and Gao, Rui and Tarasov, Andrei and Rorsman, Patrik and Zhang, Quan
Journal: iScience (2024)
Authors: Feng, Xiaogang and Andersson, Tilde and Gschwend, Julia and Fl{\"u}chter, Pascal and Berest, Ivan and Muff, Julian L and Carchidi, Daniele and Lechner, Antonie and de Tenorio, Jeshua C and Brander, Nina and others,
Journal: bioRxiv (2024): 2024--03
Authors: Zang, Shaolian and Yin, Xiaoqin and Li, Pin
Journal: Communications Biology (2023): 1297
Authors: Jin, Zihui and Zhao, Lingling and Chang, Yixin and Jin, Rongjia and Hu, Fangyu and Wu, Shuang and Xue, Zixuan and Ma, Yimeng and Chen, Chenglin and Zheng, Minghui and others,
Journal: Cell Death \& Disease (2023): 563
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