FLIPR Calcium Assay Kit
Frequently Asked Questions
What does the FLIPR Calcium Assay Kit do?
The FLIPR Calcium Assay Kit is a fast, simple and reliable fluorescence-based reagent formulation for detecting changes
in intracellular calcium in association with FLIPR. Standard protocols for evaluating changes in intracellular calcium
on FLIPR require tedious wash steps which are damaging to cell responsiveness and assay precision. In contrast, the
proprietary mechanism of action of the FLIPR Calcium Assay Kit enables the elimination of these time-consuming wash
steps. The Kit provides a Mix and Read assay format for measuring changes in intracellular calcium on FLIPR.
What does the FLIPR Calcium Assay Kit contain?
Each FLIPR Calcium Assay Kit contains the following components and is sufficient for one hundred 96-well or 384-well
plates:
- 1 bottle of 10X Reagent Buffer, Component B (10X Hanks BSS with 20 mM HEPES, pH 6.0)
- 10 vials of FLIPR Calcium Assay Reagent, Component A
- Each vial is sufficient for ten 96- or 384-well plates
Are any additional reagents required for use with the FLIPR Calcium Assay Kit?
The FLIPR Calcium Assay Kit has been developed to provide the user with all the reagents necessary to conduct a standard
calcium assay. You will need to provide 0.1N NaOH to adjust the pH to 7.4 and may find that 0.1N HCl useful for final
adjustment. Some cell types express the anion-exchange protein and for these cell types we do recommend the addition
of probenecid to the loading buffer. Probenecid is not provided with the kit.
What dye is used in the FLIPR Calcium Assay Kit?
The contents of the FLIPR Calcium Assay Kit are optimized to give a robust result when used according to the protocol
and read using the FLIPR system. Results shown above illustrate comparisons between the FLIPR Calcium Assay Kit and
fluo-3 or fluo-4. The composition of the kit is proprietary and the subject of pending patents by Molecular Devices
Corporation.
What is the excitation wavelength of the dye? Do I need additional emission filters?
In keeping with the current FLIPR optics, the dye is excited at the 488 nm wavelength of the argon ion laser and the
standard 510-570 nm emission filter is used. Additional emission filters are not required for the use of the FLIPR
Calcium Assay Kit.
Which plate format is the Calcium Assay Kit compatible with?
The Calcium Assay Kit has been successfully tested in both 96- and 384-well plate format.
What cell types can be used with the FLIPR Calcium Assay Kit?
Over 100 mammalian cell lines have been successfully used in the FLIPR system with the standard calcium assay protocol.
Several adherent and non-adherent cell types have been successfully assayed using the FLIPR Calcium Assay Kit, and
comparable results are expected with other cell lines. In many cases, use of the FLIPR Calcium Assay Kit has provided
improved data quality relative to that obtained using the conventional protocol.
Do I have to use poly-D-lysine coated plates when using the FLIPR Calcium Assay Kit?
With conventional calcium assay protocols, it is often necessary to seed mildly adherent or suspension cells into
poly-D-lysine coated plates to improve adherence and minimize cellular blow-off. Depending on the individual cell
line, with the FLIPR Calcium Assay Kit, poly-D-lysine coated plates may no longer be required. With HEK293 cells,
we have comparable results from cells seeded in regular plates and poly-D-lysine coated plates when using the FLIPR
Calcium Assay Kit. However, this may not hold true for all cell types. We therefore recommend optimization of the
assay conditions for individual cell types.
Do I need to add BSA to the assay buffer?
We recommend that all users of the FLIPR Calcium Assay Kit adhere to the protocol provided with the kit that describes
the optimal assay method. We do not add BSA to the assay buffer and have not observed any decrease in the efficiency
of dye loading by doing so. If you find BSA is required in the course of optimization of the assay then it should not
affect the mechanism of action of the kit components.
Do I need to aspirate growth media from the cell wells prior to adding the Calcium Assay Reagent?
No. The optimal protocol, as provided with the FLIPR Calcium Assay Kit, assumes no removal of growth media from the
cells. The FLIPR Calcium Assay Kit was developed and optimized to allow the direct addition of the reconstituted
reagents to the cell wells, so obtaining a 1:1 dilution of reagent in growth medium.
Can I batch load plates and run them sequentially without staggering the addition of the Calcium Assay Reagent to the plates?
With the FLIPR Calcium Assay Kit, plates can be batch loaded and then read sequentially on the FLIPR system without
variation in signal between plates. We have evidence to support that plates can be batch loaded and read over a 4-hour
period without loss of consistency. However we do stress that this may be cell line dependent and therefore recommend
that this be tested and optimized for each cell line.
At what temperature should the dye loading be conducted? Can I load the cells at room temperature? For how long should the cells be loaded with the FLIPR Calcium Assay Kit reagent?
The optimal protocol provided with the FLIPR Calcium Assay Kit recommends dye loading at 37 °C for 1 hour. However,
we do have evidence to support the successful dye loading of cells at room temperature. The length of time for dye
loading will vary depending upon cell type. Dye loading typically takes between 20 minutes and one hour; we would
recommend optimizing the conditions for each cell line.
Can I freeze any remaining 1x Loading Buffer at the end of day for use at a later date?
The 1x Loading Buffer may be frozen and stored at -20 °C for up to two weeks. However if your cells require
probenecid, it is recommended not to store frozen aliquots of Loading Buffer with probenecid and to always add fresh
probenecid on the day of the experiment.
How long is the Calcium Assay Reagent stable?
On receipt of the kit, it is recommended that the reagent buffer be removed and stored at room temperature. The FLIPR
Calcium Assay Reagent (Component A) should be stored at -20 °C. Under these conditions the reagents are stable for
six months in the original packaging. After formulation, the 1X Loading Buffer is stable for up to eight hours at room
temperature. Aliquots can be frozen and stored up to two weeks without loss of activity.
How comparable are the results obtained with the FLIPR Calcium Assay Kit to those from standard protocols using the conventional fluo-3 and fluo-4 dyes?
The results obtained with the FLIPR Calcium Assay Kit are highly comparable to those obtained from the conventional
assay protocols. The maximum signal obtained with the assay kit is usually equal to or higher than the responses
seen with fluo-3 for most cell types and comparable to that achieved with fluo-4.
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