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Calcein Red, AM

Cat. No. M10356
Calcein Red, AM Structure
Size Price Availability Quantity
1mg USD 550  USD550 In stock
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Quality Control
  • Current batch:
  • Purity Biological Stain
  • COA
  • MSDS
Biological Activity

Calcein AM is one of the most popular fluorescent probes used for labeling and monitoring cellular functions of live cells. 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.


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.
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.

PREPARATION OF WORKING SOLUTION

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.

SAMPLE EXPERIMENTAL PROTOCOL

  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.

EXAMPLE DATA ANALYSIS AND FIGURES


Figure 1. Images of Live HeLa cells stained with Calcein Red, AM (Cat# M10356). Cell nuclei were stained with Hoechst 33342 (Blue, Cat# M5112).


Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)
Species Mouse Rat Rabbit Guinea pig Hamster Dog
Weight (kg) 0.02 0.15 1.8 0.4 0.08 10
Body Surface Area (m2) 0.007 0.025 0.15 0.05 0.02 0.5
Km factor 3 6 12 8 5 20
Animal A (mg/kg) = Animal B (mg/kg) multiplied by  Animal B Km
Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

Chemical Information
Molecular Weight 1015.79
Formula
CAS Number
Purity Biological Stain
Solubility DMSO 1~5mM
Storage -20°C, protect from light, dry, sealed
References

[1] Nienke R Wevers, et al. Fluids Barriers CNS. A perfused human blood-brain barrier on-a-chip for high-throughput assessment of barrier function and antibody transport

[2] Hongli Li, et al. Toxicol Appl Pharmacol. Astragaloside IV protects blood-brain barrier integrity from LPS-induced disruption via activating Nrf2 antioxidant signaling pathway in mice

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