Protocol: Using Pluronic® F-127 to Solubilize Dyes for Cell Loading

Pluronic® F-127 is a nonionic detergent useful for solubilizing hydrophobic molecules in aqueous solutions. Pluronic® F-127 is commonly used to solubilize hydrophobic AM ester forms of fluorescent indicator dyes for calcium and other ions for loading of the dyes into cells. It also can be used to solubilize other hydrophobic dyes or compounds to facilitate cell penetration.

Preparing and handling Pluronic® F-127 stock solutions

• Pluronic® F-127 is soluble in water at 10% (w/v) or in DMSO at 20% (w/v). Heating may be necessary to achieve these concentrations.
• Store Pluronic® F-127 solutions at room temperature. DO NOT refrigerate or freeze solutions.
• Pluronic® F-127 solutions may solidify into a gel during storage, especially at low temperature. This is normal and does not affect the product, but the solution must be in liquid form before use. Heat the vial to 50-65°C for 5-10 minutes and vortex periodically until it has formed a clear liquid.

Solubilizing dyes for cell loading using Pluronic® F-127

The following is a typical procedure for loading AM esters of ion indicators into cells. Optimal conditions for cell loading may vary for different cell types or compounds.

1. Dissolve the dye or AM ester in anhydrous DMSO at 1-5 mM (or ~1000X to 500X the final desired dye concentration).
2. Immediately before use, mix equal volumes of the dye or AM ester stock solution and Pluronic® F-127, 20% in DMSO in a microcentrifuge tube.
3. Add the solution from step 2 to cell culture medium or buffer to achieve a final AM ester concentration of 1 uM to 10 uM.
4. Remove culture medium from the cells and add the medium or buffer containing diluted dye or AM ester with Pluronic® F-127.
5. Incubate cells at room temperature or 37°C for 10 minutes to 1 hour or longer.
   Note: Loading cells with indicator at room temperature may reduce dye compartmentalization into mitochondria.

Ordering Information