Membrane Potential Assay Kit

Troubleshooting Guide

1. Drop in fluorescence

When incubating cells for long time periods (e.g., over one hour), you might experience large drops (5-10K fluorescent units) on compound addition on the FLIPR system. This drop is most likely due to blowing cells off the bottom of the plate. Optimize your assay by shortening incubation times, plating cells on poly-D-lysine coated plates, and/or slowing FLIPR addition speeds.

2. Increase in fluorescence

You may observe an increase of fluorescence upon buffer only challenge. This increase is most likely due to a response of endogenous ion channels to increasing ionic strength. Patch clamp data supports this observed change. The choice of cells and expression levels of endogenous channels can greatly influence resting and changing membrane potentials.

3. No response

If you do not see a change in fluorescence, the assay needs to be optimized. Recommendations include: longer incubation and assay times, and choosing a different buffer, such as Tyrode's or specific ion-free buffers. For example, if studying a calcium channel, dye load cells using a calcium-free buffer and prepare your compound plate using a calcium containing buffer.

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