CytoCalcein™ Violet 660, AM *Excited at 405 nm*

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

1. Prepare a 2 to 5 mM stock solution of CytoCalcein™ Violet 660 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.

1. Prepare a CytoCalcein™ Violet 660 working solution of 1 to 10 µM in the buffer of your choice (e.g., Hanks and Hepes buffer). For most cell lines, CytoCalcein™ Violet 660 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 CytoCalcein™ Violet 660 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 violet laser and 675/30 nm filter set, a flow cytometer equipped with a violet laser and a 660/20 nm filter, or a fluorescence plate reader at Ex/Em = 405/660 nm cutoff 630 nm.