Many fluorescent dyes are poorly soluble in water and difficult to load into cells. In addition, organic-anion transporters located in the cell membrane can extrude dyes and indicators and thereby contribute to poor dye retention. The consequences of these features is poor performance with high background fluorescence and reduced signal to noise levels.
Figure 1: Mechanism of dye loading and retention. The uptake of fluorescent dyes into a cell is enhanced when PowerLoad Concentrate is mixed with the dye prior to addition. As the fluorescent dye is transferred into the cell, the AM ester portion of the dye is removed by cellular esterases. The dye can be shuttled out of the cell via anion transporters in the membrane. Probenecid blocks this extracellular transport thereby retaining dyes inside the cell.
To improve dye loading, Invitrogen has developed PowerLoad Concentrate, an optimized formulation of nonionic, Pluronic surfactant polyols designed to aid the solubilization of water-insoluble dyes and other materials in physiological media (Figure 2).
Figure 2: Comparison of Fluo-4 AM calcium mobilization signal window without cell loading reagent (Control), using Pluronic F-127 and PowerLoad in CHO M1 cells stimulated by carbachol.
For dye retention issues, the use of probenecid to suppress efflux of fluorescent dyes is a favorable method for reducing baseline fluorescence. To improve the utilization of this compound, we have developed a water soluble probenecid formulation (Figure 3).
Figure 3: Inhibition of dye extrusion using 2.5 mM probenecid. CHO M1 cells were incubated in dye loading solution for 60 minutes at 37° C, which accentuates the probenecid effect. Calcium release was triggered in CHO M1 cells using 20 nM carbachol. The averages of four measurements (without baseline subtraction) in a Fluo-4 NW assay were plotted.
Used together, PowerLoad Concentrate and water soluble Probenecid aid in AM ester dye-loading and retention in cells that actively extrude the de-acetylated form through anion pumps. Together, these reagents allow for maximal loading of dyes with minimal effort in imaging and high throughput screening (HTS) applications. Appropriate controls should be performed to make certain that PowerLoad concentrate is not altering the membrane properties of the cell.
For Research Use Only. Not for use in diagnostic procedures.