Coumarin 6

Staining of tumor cells by Coumarin 6
This protocol only provides a guideline, and should be modified according to your specific needs
1. Stock and dilute solution preparation for Coumarin 6 staining of cells
(1) A 60μM Coumarin 6 stock solution was obtained by dissolving Coumarin 6 powder in absolute ethanol resulting in a clear, green fluorescent solution. Dilute the Coumarin 6 stock solution (60μM) with absolute ethanol to a working solution with a final concentration of 1μM.
NOTE: The working solution must be prepared fresh on the day of the experiment.
(2) Complex formation between Coumarin 6 and β-cyclodextrin
Ethanolic Coumarin 6 (working solution) and aqueous β-cyclodextrin was mixed in varying amounts to obtain the desired Coumarin 6/β-cyclodextrin concentration ratios. The mixture was stirred for 10min at room temperature and this procedure gave a clear, highly green fluorescent solution in the case the β-cyclodextrin concentration exceeded 20μM. To stain cells the solution was added to the growing buffer for a final concentration of 30nM for the dye.
2. Cell staining and epifluorescent images
(1) Bladder cancer cells were grown in chambered cover slides in MEM medium at 37°C and 5% CO₂.
(2) For imaging the medium was replaced by phosphate buffered saline (137mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄, 1.76mM KH₂PO₄, pH 7.4) containing the Coumarin 6/β-cyclodextrin complex at a final concentration of 30nM for Coumarin 6.
(3) Epifluorescence images were taken after 10min incubation, appropriate filter settings and ×10 magnifications.
3. Experimental Precautions
(1) Coumarin 6 solution should be stored in the dark throughout the experiment.
(2) This protocol and all reagents are for scientific research use only.

References:
[1] Edetsberger M, Knapp M, Gaubitzer E, et al. Effective staining of tumor cells by coumarin-6 depends on the stoichiometry of cyclodextrin complex formation[J]. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 2011, 70(3): 327-331.