Calcein Red AM, a red-emitting derivative of the green fluorophore calcein AM, is a cell-permeable, non-fluorescent acetoxymethyl ester compound commonly utilized to assess cell viability and cytotoxicity. following intracellular esterase cleavage, the fluorescent product is retained in live cells and exhibits characteristic excitation and emission peaks at 560 nm and 574 nm, respectively.

I. Preparation of Calcein Red AM Working Solution
1. Preparation of Stock Solution: DMSO can be used to prepare a 5-10 mM stock solution for future use.
2. Preparation of Working Solution: Dilute the stock solution with prewarmed pure DMEM or PBS to prepare a 1-10 μM Calcein Red AM working solution for future use.
Notes:
1) Adjust the concentration of the Calcein Red AM working solution according to actual needs and prepare it immediately before use.
2) Store unused stock solution at -80°C or -20°C, aliquot, and store in a dark place. Avoid repeated freeze-thaw cycles.
II. Cell Staining
1. Suspension Cells
1) Collect cells by centrifugation and wash twice with PBS for 5 minutes each. The cell density should be 1×10^6/mL.
2) Add 1 mL of Calcein Red AM working solution and incubate at 37°C for 30-45 minutes.
3) Centrifuge at 400 g for 3-4 minutes and discard the supernatant.
4) Add PBS Wash the cells twice, 5 minutes each.
5) Resuspend the cells in 1 mL of pure DMEM or PBS and observe using a fluorescence microscope or flow cytometer.
2. Adherent Cells
1) Plate the adherent cells on a sterile coverslip.
2) Remove the coverslip from the culture medium and aspirate the excess medium.
3) Add 100 μL of the dye working solution, gently shake to completely cover the cells, and incubate at 37°C for 30-45 minutes.
4) Aspirate the dye working solution, wash the cells 2-3 times with culture medium, 5 minutes each, and observe using a fluorescence microscope or fluorescence microscopy reader.

Precautions
1. Please adjust the concentration of the Calcein Red AM working solution and incubation time according to your specific needs.
2. This product is for professional scientific research use only and is not intended for clinical diagnosis or other purposes.
3. For your safety and well-being, please wear a lab coat and disposable gloves when handling.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.