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CAS:168482-84-6
Storage:Powder:-20℃，2 years;Insolvent(Mother Liquid):-20℃，6 months;-80℃，1 year
Purity:≥95%
Appearance:Solid
Manual Download
Calcein Blue AM is a cell permeable Calcein Blue. Upon entry into living cells, the weakly fluorescent Calcein Blue AM is hydrolyzed to Calcein Blue, which has an excitation/emission maximum similar to DAPI, Hoechst, and AMCA. This special spectral separation from typical green and red fluorophores, such as FITC, TMR, and Texas red, provides an additional option for multiplex experiments. Since Calcein Blue AM is inherently fluorescent, appropriate filter Settings and additional washing steps may be required to minimize background fluorescence.
Flow cytometer (for reference only)
Excitation 350 nm or 405 nm laser
Emission 450/40 nm filter
Instrument specification(s) Pacific Blue channel
Fluorescence microscope (for reference only)
Excitation DAPI filter set
Emission DAPI filter set
Recommended plate Black wall/clear bottom
Fluorescence microplate reader (for reference only)
Excitation 360
Emission 450
Cutoff 420
Recommended plate Black wall/clear bottom
Instrument specification(s) Bottom read mode
Example Protocol (for reference only)
Calcein Blue, AM Stock Solution
Prepare a 2 to 5 mM stock solution of Calcein Blue AM in high-quality, anhydrous DMSO.
If it is necessary to prepare a stock solution, it is recommended to store in aliquots to avoid product failure caused by repeated freezing and thawing.
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%.
Calcein Blue, AM Working Solution
Prepare a Calcein Blue 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 Blue 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 air de-esterified indicators.
SAMPLE EXPERIMENTAL Protocol (for reference only)
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 Blue AM working solution to the culture.
Incubate cells at 37 ℃ 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 DAPI filter set, a flow cytometer equipped with a 450/40 nm filter (Pacific Blue channel), or a fluorescence plate reader at Ex/Em = 360/450 nm cutoff 420 nm.
