Membrane Potential Assay Kit
Frequently Asked Questions
What does the FLIPR Membrane Potential Assay Kit do?
The kit detects changes in membrane potential due to ion flux across the cellular membrane. Eliminates extensive
pre-assay and assay steps, thus, eliminating experimental variability.
What does the FLIPR Membrane Potential Assay Kit contain?
Each FLIPR Membrane Potential Assay Kit contains the following components and is sufficient for one hundred 96-well
or 384-well microplates:
- 1 bottle of 10X Reagent Buffer, Component B (10X Hanks' BSS with 200mM HEPES, pH6).
- 10 vials of FLIPR Membrane Potential Assay Reagent, Component A.
- Each vial is sufficient to assay ten 96- or 384-microwell plates.
What dye is used in the FLIPR Membrane Potential Assay Kit?
The composition of kit is subject of a patent application and is proprietary. Results obtained with the kit are much
faster and robust when compared to the standard protocol for measuring membrane potential in FLIPR.
Are any additional reagents required for use with the FLIPR Membrane Potential Assay Kit?
You will need 1.0N NaOH and 1.0N HCl to adjust the pH of the 1X Assay Buffer to 7.4. Use of probenecid is not
recommended. Control of "leaky" ion channels or pumps can require the use of appropriate blockers. Several of these
blockers have been tested with the FLIPR Membrane Potential Assay Kit and found to be quite compatible. Blockers
tested include: barium chloride as a blocker of voltage-gated potassium channels; suramin and pyridoxal-5-phosphate
as blockers of P2X channels; TTX as a blocker of voltage-gated sodium channel.
What is the excitation wavelength of the dye? Do I need additional emission filters?
Dye is excited at 488 nm wavelength of the argon ion laser. An additional emission filter (cat #0310-4077) is
required for the use of the FLIPR Membrane Potential Assay Kit.
Which plate format is the Membrane Potential Assay Kit compatible with?
The kit has been successfully tested in both 96- and 384-well formats.
What cell types can be used with the FLIPR Membrane Potential Assay Kit?
Several cell types have been successfully tested, including primary cells, mouse fibroblasts, PC-12, THP-1, 1321N1 astrocytoma cells, and CHO and HEK293 cells over-expressing various ion channels.
Do I have to use poly-D-lysine coated plates?
Coated plates are used to minimize cellular blowoff from washing and FLIPR additions. The FLIPR Membrane Potential Assay Kit is a true mix-and-read assay, thus removing the need for coated plates. However, this may not hold true for all cell types, therefore, assay conditions should be optimized for each cell type. The use of coated plates is recommended if fluorescent counts drop significantly (5K) upon FLIPR addition when using non-coated plates.
Do I need to aspirate growth media from the cell wells prior to adding the Membrane Potential Assay Reagent?
No. If you do choose to aspirate growth medium, please maintain the volumes described in the protocol by backfilling the wells with an equal volume of buffer. Some cells, ion channels, and compounds are serum sensitive, so growth medium removal is necessary.
Can I batch load plates? How long should cells be loaded?
Yes. Data supports dye loading for 15 minutes up to 1 hour and can increase throughput as much as 10 times when compared
to the standard membrane potential assay. Each cell type must be tested and optimized for dye loading time.
At what temperature should the dye loading be conducted?
Dye loading is generally performed at 37 °C, but data supports dye loading at room temperature.
Can I freeze any remaining Loading Buffer at the end of day for use at a later date?
Yes. Loading Buffer may be stored frozen for up to two weeks. Warm up Loading Buffer in a 37 °C water bath and
vortex to ensure complete solubility prior to loading cells.
How long is the Membrane Potential Assay Reagent stable?
All kit components are stable for 6 months from date of shipment when stored at room temperature in the original packaging.
How comparable are the results obtained with the FLIPR Membrane Potential Assay Kit to the standard protocol using DiBAC?
Data suggests the Membrane Potential Assay Kit is far superior; the dye is much faster, the signal-to-background is
higher, and the throughput can be increased ten-fold. For example, membrane depolarization can be detected with the
kit in less than 10 seconds. In addition, data can be obtained using the FLIPR Membrane Potential Assay Kit where
equivalent data is unobtainable with the standard protocol, especially small changes in membrane potential.
|
