Calcein UltraGreen™ AM
![Fixability of live HeLa cells stained with Calcein AM and Calcein UltraGreen AM. HeLa cells were stained with Calcein AM (Catalog #22004) and Calcein UltraGreen AM (Catalog #21905) and then fixed in 4% formaldehyde. Image acquisition was performed on a fluorescence microscope equipped with a FITC filter set. Notably, staining achieved with Calcein UltraGreen AM demonstrated prolonged stability post-fixation, indicating its effectiveness for long-term cellular imaging applications.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalcein-ultragreen-am%2Ffigure-for-calcein-ultragreen-am_s5VRi.png&w=640&q=75)
![Fixability of live HeLa cells stained with Calcein AM and Calcein UltraGreen AM. HeLa cells were stained with Calcein AM (Catalog #22004) and Calcein UltraGreen AM (Catalog #21905) and then fixed in 4% formaldehyde. Image acquisition was performed on a fluorescence microscope equipped with a FITC filter set. Notably, staining achieved with Calcein UltraGreen AM demonstrated prolonged stability post-fixation, indicating its effectiveness for long-term cellular imaging applications.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalcein-ultragreen-am%2Ffigure-for-calcein-ultragreen-am_s5VRi.png&w=640&q=75)
![Fixability of live HeLa cells stained with Calcein AM and Calcein UltraGreen AM. HeLa cells were stained with Calcein AM (Catalog #22004) and Calcein UltraGreen AM (Catalog #21905) and then fixed in 4% formaldehyde. Image acquisition was performed on a fluorescence microscope equipped with a FITC filter set. Notably, staining achieved with Calcein UltraGreen AM demonstrated prolonged stability post-fixation, indicating its effectiveness for long-term cellular imaging applications.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalcein-ultragreen-am%2Ffigure-for-calcein-ultragreen-am_s5VRi.png&w=128&q=25)
![Fluorescence images of HeLa cells stained with Calcein UltraGreen™ AM (upper row) or Calcein AM (lower row) in a Costar black wall/clear bottom 96-well plate. After washing, growth media were added back, and the cells were monitored using a microscope with FITC filter for up to 24 hours.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalcein-ultragreen-am%2Ffigure-for-calcein-ultragreen-am_QBRmM.jpg&w=128&q=25)
![Images of Live HeLa cells stained with Calcein UltraGreen™, AM (Cat.21905). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalcein-ultragreen-am%2Ffigure-for-calcein-ultragreen-am_qSPAu.jpg&w=128&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 Calcein UltraGreen™ AM in high-quality, anhydrous DMSO.
Note: When reconstituted in DMSO, Calcein UltraGreen™ AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
Prepare a Calcein UltraGreen™ AM working solution of 1 to 10 µM in the buffer of your choice (e.g., Hanks and Hepes buffer). For most cell lines, Calcein UltraGreen™ AM 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: 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.
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. A variety of ReadiUse™ Probenecid products, Including water-soluble, sodium salt, and stabilized solutions, can be purchased from AAT Bioquest.
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 Calcein UltraGreen™ AM 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 FITC filter set, a flow cytometer equipped with a blue laser and a 530/30 nm filter (FITC channel), or a fluorescence plate reader at Ex/Em = 490/525 nm cutoff 515 nm.
Name | Excitation (nm) | Emission (nm) |
Calcein Red™ AM | 562 | 576 |
Calcein UltraBlue™ AM | 359 | 458 |
Calcein Blue, AM *CAS 168482-84-6* | 354 | 441 |
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