AAT Bioquest

Calcein Red™ AM

Images of Live HeLa cells stained with Calcein Red <sup>TM</sup>, AM (Cat.21900). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).
Images of Live HeLa cells stained with Calcein Red <sup>TM</sup>, AM (Cat.21900). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).
Images of Live HeLa cells stained with Calcein Red <sup>TM</sup>, AM (Cat.21900). Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17535).
Images of HeLa cells stained with Calcein Red&trade; AM. Left: Live HeLa cells; Right: Fixed HeLa cells.
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Physical properties
Molecular weight1015.79
Spectral properties
Excitation (nm)562
Emission (nm)576
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Excitation (nm)
Emission (nm)
Calcein AM is one of the most popular fluorescent probes used for labeling and monitoring cellular functions of live cells. However, the single color of Calcein AM makes it impossible to use this valuable reagent in the multicolor applications. For example, it is impossible to use Calcein AM in combination of GFP-tranfacted cells due to the same color to GFP. To address this color limitation of Calcein AM, we have developed Calcein Orange™, Calcein Red™ and Calcein Deep Red™. These new Calcein AM analogs enable the multicolor labeling and functional analysis of live cells in combination with Calcein AM. Non-fluorescent Calcein Red™ AM can easily get into live cells and hydrolyzes to generate strongly fluorescent Calcein Red™ dye. Calcein Red™ dye can be monitored with the common TRITC/Cy3 filter set. AAT Bioquest offers Calcein Red™ as a reference dye to Calcein Red™ AM.


Flow cytometer

Excitation532/561 nm laser
Emission585/40 nm filter

Fluorescence microscope

ExcitationTRITC filter set
EmissionTRITC filter set
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Example protocol


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.

Calcein Red™ AM Stock Solution
Prepare a 2 to 5 mM stock solution of Calcein Red™ AM 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.


Calcein Red™ AM Working Solution
Prepare a Calcein Red™ 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 Red™ 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     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.


  1. Prepare cells for imaging.
  2. 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.
  3. Add Calcein Red™ AM working solution to the culture.
  4. Incubate cells at 37 °C for 30 to 60 minutes.
  5. 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.
  6. Measure the fluorescence intensity using either a fluorescence microscope equipped with a TRITC filter set, a flow cytometer equipped with green/yellow laser and a 585/40 nm filter, or a fluorescence plate reader at Ex/Em = 540/590 nm cutoff 570 nm. 


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Calcein Red™ 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 mg0.5 mg1 mg5 mg10 mg
1 mM98.446 µL492.228 µL984.455 µL4.922 mL9.845 mL
5 mM19.689 µL98.446 µL196.891 µL984.455 µL1.969 mL
10 mM9.845 µL49.223 µL98.446 µL492.228 µL984.455 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles


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Spectral properties

Excitation (nm)562
Emission (nm)576



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