CytoCalcein™ Violet 660, AM *Excited at 405 nm*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Molecular weight | 812.87 |
Solvent | DMSO |
Excitation (nm) | 402 |
Emission (nm) | 660 |
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 |
Overview | SDSProtocol |
Molecular weight 812.87 | Excitation (nm) 402 | Emission (nm) 660 |
Platform
Flow cytometer
Excitation | 405 nm laser |
Emission | 660/20 nm filter |
Fluorescence microscope
Excitation | 405 nm laser |
Emission | 675/30 nm |
Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
Excitation | 405 |
Emission | 660 |
Cutoff | 630 |
Recommended plate | Solid black |
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 CytoCalcein™ Violet 660 in high-quality, anhydrous DMSO.
Note: The nonionic detergent Pluronic® F-127 can be used to increase the aqueous solubility of AM esters. In the staining buffer, the final Pluronic® F-127 concentration should be approximately 0.02%. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest. Avoid long-term storage of AM esters in the presence of Pluronic® F-127.
PREPARATION OF WORKING SOLUTION
Prepare a CytoCalcein™ Violet 660 working solution of 1 to 10 µM in the buffer of your choice (e.g., Hanks and Hepes buffer). For most cell lines, CytoCalcein™ Violet 660 at the final concentration of 4 to 5 µM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note: If your cells contain organic anion transporters, probenecid (1–2.5 mM) or sulfinpyrazone (0.1–0.25 mM) may be added to the working solution to reduce leakage of the de-esterified indicators.
SAMPLE EXPERIMENTAL PROTOCOL
Prepare cells for imaging.
Remove the cell culture medium and wash cells once with serum-free buffer to remove any remaining media.
Note: Serum in cell culture media may contain esterase activity, which can increase background interference.
Add CytoCalcein™ Violet 660 working solution to the culture.
Incubate cells at 37 °C for 30 to 60 minutes.
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.
Measure the fluorescence intensity using either a fluorescence microscope equipped with a violet laser and 675/30 nm filter set, a flow cytometer equipped with a violet laser and a 660/20 nm filter, or a fluorescence plate reader at Ex/Em = 405/660 nm cutoff 630 nm.
Calculators
Common stock solution preparation
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 123.021 µL | 615.105 µL | 1.23 mL | 6.151 mL | 12.302 mL |
5 mM | 24.604 µL | 123.021 µL | 246.042 µL | 1.23 mL | 2.46 mL |
10 mM | 12.302 µL | 61.51 µL | 123.021 µL | 615.105 µL | 1.23 mL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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References
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Journal: bioRxiv : the preprint server for biology (2023)
Authors: Wichai, Uthai and Keawsomnuk, Ploychanok and Thongin, Saowarose and Mukthung, Chaiyot and Boonthip, Chatchai and Pittayakhajonwut, Pattama and Ketsawatsomkron, Pimonrat and Bunyapraphatsara, Nuntavan and Muta, Kenjiro
Journal: BMC complementary medicine and therapies (2023): 18
Authors: Won, Jun Pil and Kim, Eunsu and Hur, Jinwoo and Lee, Hyuk Gyoon and Lee, Won Jin and Seo, Han Geuk
Journal: Journal of ethnopharmacology (2023): 116267
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