FLIPR Calcium 3 Assay Kit

Assay Protocol

• About the FLIPR Calcium 3 Assay Kit
• Kit contents
• Additional materials needed but not included in kit
• Storage information
• Cell handling
• Preparation of loading buffer
• Loading the cells
• Running the FLIPR Calcium 3 assay on FLIPR
• Running the FLIPR Calcium 3 assay on FlexStation
• Representative data analysis for FLIPR
• Representative data analysis for FlexStation
• Product use limitations and warranty

Introduction

about the FLIPR Calcium 3 Assay Kit

The FLIPR Calcium 3 Assay Kit from Molecular Devices Corporation provides a fast, simple and reliable fluorescence-based assay for detecting changes in intracellular calcium. With this kit, calcium assays on FLIPR or FlexStation™ become a mix-and-read procedure in which cells are incubated with the kit reagents for one hour and transferred directly to FLIPR or FlexStation for evaluation. There are no intermediate wash steps involved.

Conventional protocols for evaluating changes in intracellular calcium with FLIPR and FlexStation are multi-step procedures consisting of a dye-loading step with Fluo3, Fluo4, or other calcium indicators followed by extensive washing of the cells prior to running the assay. Several problems are routinely encountered when washing dye-loaded cells, all of which add to experimental variability.

Problems in a conventional assay protocol include:

• Cells removed from the plates during the washing procedure
• Reduced responsiveness (competence) of cells after washing
• Spontaneous calcium flux in the negative control cells upon buffer addition
• Variation in the residual volume of wash buffer, leading to variation in the concentration of test compound
• Incomplete washing, resulting in a significant drop in signal upon addition of test compound

Molecular Devices has developed the FLIPR Calcium 3 Assay Kit, which not only eliminates the cause of variability in the data and reduces the number of steps in the conventional wash protocol using Fluo-3 or Fluo-4, but also offers additional advantages over the FLIPR Calcium and Calcium PLUS Assay Kits.

Advantages of the FLIPR Calcium 3 Assay Kit include:

• Enhancement of signal dynamic change
• Improvement of data quality
• Reduced well-to-well variation
• Ease of use with both adherent and non-adherent cells
• Rapid procedure with less hands-on time
• Fewer steps in the assay resulting in higher sample throughput
• Minimal perturbation of the cells reducing spontaneous calcium fluxes
• Broad range of applications for GPCR targets and calcium channels

Applications

The kit provides a homogeneous assay for calcium flux. It is designed to work for the majority of GPCRs, including chemokine receptors and other difficult targets, and calcium channels.

Materials & equipment

kit components

Each kit contains the following components.

R8091 (Explorer Kit)		10 vials Component A 1 bottle Component B (ready to use HBSS buffer – 1X Hank's Balanced Salt solution with 20 mM Hepes buffer, pH 7.4). Sufficient for ten 96- or 384-well plates. Each vial is sufficient for assaying one 96- or 384-well plate.
R8090 (Bulk Kit)		10 vials, sufficient for one hundred 96- or 384-well plates. Each vial is sufficient for assaying ten 96- or 384-well plates.
R8108 (Express Kit)		2 vials, sufficient for one hundred 96-well or 384-well plates. Each vial is sufficient for assaying fifty 96- or 384-well plates.

Additional materials needed but not included in kit

HBSS Buffer (1X Hank's Balanced Salt solution with 20 mM Hepes buffer)		10X Hank's Balanced Salt Solution (#14065-056, Gibco or equivalent) 1M Hepes buffer solution (#9319, Irvine Scientific or equivalent) Water for cell culture (# 9312, Irvine Scientific or equivalent)
Probenecid (inhibitor for the anion-exchange protein) may be required with some cell lines.		Sigma or other chemical suppliers

Storage & handling

On receipt of the kit, store the FLIPR Calcium 3 Assay Kit at -20 °C. Under these conditions the reagents are stable for six months in the original packaging.

After formulation, the Loading Buffer is stable for up to eight hours at room temperature. Aliquots can be frozen and stored for up to 5 days without loss of activity.

FLIPR Calcium 3 Assay Kit experimental protocol

cell handling

The FLIPR Calcium 3 Assay Kit is designed to work with many cell types, both adherent and non-adherent. In this section we provide guidelines on how to set up the cells for use with the assay kit.

We recognize that a variety of cell handling conditions might be adopted at the discretion of the user based on standard operating procedures in the laboratory. Optimal cell conditions for the FLIPR Calcium 3 Assay Kit require the creation of a confluent cell monolayer before placing the plates in FLIPR or FlexStation. In general, we recommend starting with 80,000 cells/well for a 96-well plate and 20,000 cells/well for a 384-well plate.

• For adherent cells, we recommend seeding cells overnight with a plating volume of 100 µl/well for 96-well plates or 25 µl/well for 384-well plates.
• For non-adherent cells, we recommend centrifugation of the cells from culture medium and suspension of the pellet in culture medium. Add 100 µl (96-well plate) or 25 µl (384-well plate) of cell suspension to each well of the plate. It is recommended that you then centrifuge the plates at 1000 rpm for up to 4 minutes (brake off).

1. Preparation of loading buffer

The following procedure is designed for preparation of the loading buffer per vial of the Explorer Kit (R8091), the Bulk Kit (R8091), or the Express Kit (R8108).

1. To prepare the 1X HBSS Buffer (for the Bulk Kit and the Express Kit only, Explorer Kit contains ready to use HBSS buffer component B) pipette 100 ml of 10X Hank's Balanced Salt Solution and 20 ml of 1M Hepes buffer solution to 880 ml cell culture treated water.
2. Remove one vial of FLIPR Calcium 3 Assay reagent and equilibrate to room temperature.
3. Dissolve contents of vial completely by adding 10 ml (for Explorer kit and Bulk kit), 20 ml (for Express Kit) of 1X HBSS Buffer. Mix by repeated pipetting or vortexing until the contents are completely dissolved.
4. Prepare the Loading Buffer by diluting the vial mixture in an additional 90 ml for the Bulk Kit or 490 ml for the Express Kit of 1X HBSS Buffer. Multiple washes of the vial are necessary to completely transfer the contents.

   Note: If your cells require probenecid, then a stock solution should be prepared and added fresh to 1X HBSS buffer for preparation of the Loading Buffer at a final in-well working concentration of 2-2.5 mM. Do not store frozen aliquots of Loading Buffer with probenecid and always prepare fresh probenecid on the day of the experiment.

   Warning: the components supplied are sufficient for proper cell loading. For optimum results it is important NOT to add any additional reagents or change volumes and concentrations.

2. Loading the cells using loading buffer

1. Remove cell plates from the incubator or centrifuge. Do not remove the supernatant. Add an equal volume of Loading Buffer to each well (100 µl per well for 96-well plates, 25 µl for 384-well plates).

   Note: Although Molecular Devices does not recommend washing cells before dye loading, growth medium and serum factors can be washed away before adding the Loading Buffer, provided residual volumes after the wash step are as described. Alternatively, cells can be grown in serum-free conditions.

2. Incubate cell plates for 1 hour at 37 °C and then keep the plates at room temperature until used.

   Note: In some cases, incubation at room temperature may work better.

   Warning: do NOT wash the cells after dye loading.

3. Running the Calcium 3 assay on FLIPR

1. After incubation transfer the plates directly to FLIPR and begin the calcium assay as described in the FLIPR system manual.
2. For a signal test, a starting average count of 7,000-10,000 is recommended.
3. Recommended experimental setup parameters are as follows. Note that the addition speeds are faster than in the conventional protocol because of the increased robustness of the cells after the new loading procedure.

   Faster addition speeds can lead to better mixing of compounds and lower signal variance across the plate.

   Parameters	96-well plate	384-well plate
   Addition Volume (µl)	50	25
   Compound Concentration (Fold)	5X	3X
   Addition Speed (µl/sec) Adherent cells	50-100	10-20
   Addition Speed (µl/sec) Non-adherent cells	10-20	5-10

4. Running the Calcium 3 assay on FlexStation

1. Recommended experimental setup parameters are as follows. Set up your FlexStation using SoftMax Pro well ahead of the time you want to read the plate.

   Parameters
   Excitation Wavelength (nm)	485
   Emission Wavelength (nm)	525
   Emission Cut-off (nm)	515
   Pipette Height (µl)	230
   Transfer Volume (µl)	50
   Compound Concentration (Fold)	5X
   Addition Speed (Rate) Adherent Cells	2
   Addition Speed (Rate) Non-adherent cells	1

2. After incubation, transfer the assay plate directly to the FlexStation assay plate carriage and run the assay.
3. The assay should be complete within 3 to 5 minutes after addition, but we recommend collecting data for a minimum of 6 minutes during assay development.
4. Analyze the data using SoftMax® Pro software from Molecular Devices.

5. Representative data analysis (FLIPR)

		Figure 1. CHO cells stably transfected with muscarinic receptor 1 (CHO-M1) were seeded overnight in 100 µl per well of a 96-well Costar plate. Cells were incubated with 100 µl of the FLIPR Calcium 3 Assay Kit, Calcium Plus Assay Kit, or Fluo4 (4 mM) for 1 hour at 37 °C. Cells incubated with Fluo4 were washed 3 times with 200 µl 1X HBSS buffer per well using an Embla 96/384 well washer (Molecular Devices). Then 1X HBSS buffer (200 µl/well) was added. Carbachol was added (50 µl/well) to achieve the final indicated concentration (n =12/group). B. Data quality generated for calcium response of a GPCR cell line using the FLIPR Calcium 3 Assay Kit, the Calcium Plus Kit or Fluo-4 in a wash procedure. Each data point represents the Z' factor for a GPCR agonist at its EC75 runs in all wells of a 96-well plate.

		Figure 2. CHO cells stably transfected with CCR5 and Gqi5 were seeded overnight in 100 µl per well of a 96-well Poly-D-Lysine coated plate. The cells were incubated with 100 µl of the FLIPR Calcium 3 Kit for 1 hour at 37 °C. RANTES was added (50 µl /well) to achieve the final indicated concentration (n =4/group).

6. Representative data analysis (FlexStation)

		Figure 3. CHO cells stably transfected with CCR5 and Gaqi5 were seeded overnight in 100 µl on a 96-well Poly-D-Lysine coated plate. The cells were incubated with 100 µl of the FLIPR Calcium 3 Assay Kit for 1 hour at 37 °C. RANTES was added (50 µl/well) to achieve the final indicated concentration (n=4/group).

Product use limitations and warranty

All Molecular Devices reagent products are sold For Research Use Only. Reagents may contain chemicals that are harmful. Due care should be exercised to prevent direct human contact with the reagent.

Each product is shipped with documentation stating specifications and other technical information. Molecular Devices products are warranted to meet or exceed the stated specifications. Molecular Devices' sole obligation and the customer's sole remedy are limited to replacement of the products free of charge in the event that the product fails to perform as warranted.

Molecular Devices Corporation makes no other warranties, either expressed or implied, including without limitation the implied warranties of merchantability and fitness for a particular purpose or use.

Please note that the Calcium 3 Assay Kit is supplied as a prototype product and deviates from standard ISO procedures. Molecular Devices is continuing to evaluate the kit in order to optimize performance. While we expect this prototype kit to be representative of the final product, Molecular Devices reserves the right to modify the kit and cannot guarantee that this exact formulation will continue to be made available.

The purchase of this reagent includes a non-exclusive license to practice Bayer A.G., U.S. Patent 6,420,183 B1 and foreign counterparts thereof in conjunction with and only in conjunction with the use of this reagent purchased from Molecular Devices Corp.