The steps for applying SBFI into cells or samples is described below.

1. Cell Preparation - A stock solution of SBFI-AM is obtained by dissolving DMSO in a concentration of 10 mM.
2. Preparation of SBFI loading buffer - Pluronic F-127 is used for optimum cell loading of SBFI (because of the poor aqueous solubility of the dye). DMSO is typically mixed with an equal volume of 25% Pluroninc F-127 prior to being added to the cell loading buffer.
3. Loading SBFI into cells - Remove the buffer from the cells and incubate suspensions for 60 minutes at room temperature. 
4. Washing of the dye - Cells are then washed and bound in agarose gel, and then placed in a quartz cuvette. They are then flushed with Krebs-Henseleit buffer at a rate of 0.6 ml/min at 37° C. 
5. Fluorescence - Fluorescence can then be measured at different wavelengths with an excitation spectrofluorometer (e.g. SPEX).