FLIPR Calcium 6 and Calcium 6-QF Assay Kits 1 FLIPR® Calcium 6 Evaluation Kit Product # R8194 About the FLIPR ® Calcium 6 Evaluation Assay Kit The FLIPR® Calcium 6 Evaluation Assay Kit from Molecular Devices, LLC (hereafter referred to as Molecular Devices) offers a convenient way to compare the performance of the FLIPR® Calcium 6 Assay Kit (R8290) and the FLIPR® Calcium 6-QF Assay kit (R8192) side by side and determine the optimal kit for assay conditions, targets, cell lines, or application. If required, this kit can be compared with materials in the FLIPR® Calcium Assay Evaluation Kit (R8172), which contains FLIPR® Calcium 3, 4 and 5 formulations. The FLIPR® Calcium Assay kits provide a fast, simple, and reliable fluorescence-based assay for detecting changes in intracellular calcium. The Calcium 6 kits are based on a novel calcium indicator that provides a larger signal window. The Calcium 6 kit also uses the same proprietary quench technology used in the Calcium 4 and 5 Assay Kits. The Calcium 6-QF kit provides a quench free option, with no masking dye included in the formulation. Each kit provides mix-and-read procedures for calcium flux assays in which cells are incubated with the kit reagents for two hours and transferred directly to the FLIPR® or FlexStation® instruments for evaluation. There are no intermediate wash-steps required with these kits. TABLE OF CONTENTS INTRODUCTION Page Assay Principle 2 Applications 3 MATERIALS AND EQUIPMENT Kit Components 3 Materials required but not provided 4 Storage and handling 4 CALCIUM 6 AND CALCIUM 6-QF EXPERIMENTAL PROTOCOLS Quick Start Protocol 4 Cell handling 5 Preparation of Loading Buffer 5 Cell loading 6 Running the calcium mobilization assay 6 FLIPR® instrument settings 7 FLIPR® Tetra instrument settings (EMCCD and ICCD cameras) 8-9 FlexStation® instrument settings 9 EXAMPLE DATA 9-10 TROUBLE SHOOTING GUIDE 11-12 PRODUCT USE LIMITATIONS AND WARRANTY 13
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FLIPR Calcium 6 and Calcium 6-QF Assay Kits 1
FLIPR® Calcium 6 Evaluation Kit Product # R8194
About the FLIPR® Calcium 6 Evaluation Assay Kit The FLIPR® Calcium 6 Evaluation Assay Kit from Molecular Devices, LLC (hereafter referred to as Molecular Devices) offers a convenient way to compare the performance of the FLIPR® Calcium 6 Assay Kit (R8290) and the FLIPR® Calcium 6-QF Assay kit (R8192) side by side and determine the optimal kit for assay conditions, targets, cell lines, or application. If required, this kit can be compared with materials in the FLIPR® Calcium Assay Evaluation Kit (R8172), which contains FLIPR® Calcium 3, 4 and 5 formulations. The FLIPR® Calcium Assay kits provide a fast, simple, and reliable fluorescence-based assay for detecting changes in intracellular calcium. The Calcium 6 kits are based on a novel calcium indicator that provides a larger signal window. The Calcium 6 kit also uses the same proprietary quench technology used in the Calcium 4 and 5 Assay Kits. The Calcium 6-QF kit provides a quench free option, with no masking dye included in the formulation. Each kit provides mix-and-read procedures for calcium flux assays in which cells are incubated with the kit reagents for two hours and transferred directly to the FLIPR® or FlexStation® instruments for evaluation. There are no intermediate wash-steps required with these kits.
TABLE OF CONTENTS
INTRODUCTION Page
Assay Principle 2 Applications 3
MATERIALS AND EQUIPMENT
Kit Components 3 Materials required but not provided 4 Storage and handling 4
Reagent Description R8194 FLIPR Calcium 6 (Evaluation Kit) 3 vials Calcium 6 Explorer Kit Component A (R8190)
3 vials each Calcium 6-QF Explorer Kit Component A & Component C (R8192)
1 bottle Component B 1X Hank’s Balanced Salt solution (HBSS) plus 20 mM HEPES buffer, pH 7.4
The entire kit is sufficient for six 96-, 384-, or 1536-well plates. Each vial is sufficient for one 96-, 384-, or 1536-well plate.
Materials required but not provided
Table 2: Reagents and supplies
Item Suggested Vendor Component B*: HBSS Buffer (1X Hank’s Balanced Salt solution plus 20 mM HEPES buffer) pH 7.4 *Component B is provided for Explorer kits only.
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)
FLIPR Calcium 6 and Calcium 6-QF Assay Kits 4
Probenecid (inhibitor for the anion-exchange protein) may be required with some cell lines to insure the dye stays inside the cell and is not pumped back out. Prepare a stock solution of 500 mM in 1N NaOH, then dilute to 250 mM in HBSS buffer. Prepare loading buffer such that the final in-well concentration of probenecid is 2.5 mM when added to cells.
Sigma (# P8761) or other chemical suppliers TIP: Use of water soluble probenecid is also possible following individual manufacturer instructions TIP: With Calcium 6 Kit and Calcium 6-QF Kit, it may also be possible to run with less probenecid or none at all if the target is sensitive to probenecid. Assay development is required to determine the best concentration.
Storage and Handling
On receipt of the FLIPR® Calcium 6 Evaluation Kit, store contents at -20o 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 probenecid) without loss of activity.
Quick Start Protocol
Plate cells in microplates and incubate overnight at 37oC, 5% CO2
Prepare the loading buffer the following day
Remove cell plates from the incubator
o Calcium 6 Kit - add an equal volume of loading buffer to each well (i.e. 25 L of loading
buffer to 25 L of cells and media for a 384-well plate)
o Calcium 6 QF Kit – remove the culture media and add 25 L HBSS + 20mM HEPES
followed by 25 L dye loading buffer.
Note: Serum and other components in media will cause hydrolysis of the dye and lower the
overall signal window. While the masking dye in the Calcium 6 kit will significantly reduce
extracellular background caused by hydrolysis of dye, the Calcium 6-QF kit is quench free and
does not provide the same benefit. Removal of the culture media will prevent hydrolysis and
increased background in assays run with Calcium 6-QF.
Return plates to the incubator and incubate two hours at 37oC, 5% CO2
Prepare compound plates
Run experiment on FLIPR® or FlexStation® instruments
FLIPR Calcium 6 and Calcium 6-QF Assay Kits 5
Experimental Protocol A. Cell Handling
The FLIPR® Calcium 6 and FLIPR® Calcium 6-QF Assays are designed to work with many cell types, both
adherent and non-adherent. Standard procedures vary across laboratories and we recognize that a
variety of cell handling conditions might be adopted at the discretion of the user. In this section, we
provide general guidelines for preparing cells for use with the assay kit.
Adherent cells are the most frequently used cells with the kits. They are typically plated the day prior to
an experiment and then incubated in a 5% CO2, 37C incubator overnight. See Table 3 for suggested
plating volumes and seeding densities to create an 80-90% confluent cell monolayer before placing the
plates in the FLIPR® or FlexStation® instruments.
Table 3: Suggested plating volumes and seeding densities
For non-adherent cells, we recommend centrifuging cells from culture medium and re-suspending the pellet in culture medium or appropriate buffer of choice on the day of the experiment. Cells can be dye-loaded in a tube or while plated. It is recommended after the cells are plated, the plates be centrifuged at 100 x g for up to 4 minutes (with brake off). Alternatively, non-adherent cells can be treated like adherent cells, plating the day before the assay using the same plating volumes and seeding densities, as long as the cells are seeded onto coated plates (e.g.: poly-D-lysine or collagen) to ensure good attachment to the plate bottom.
B. Preparation of Loading Buffer
The following procedure is designed for preparation of the Calcium 6 Assay Kit Loading Buffer per vial of the Explorer Kit.
1. Remove one vial of Calcium 6 Assay Reagent (Component A) from the freezer and equilibrate to
room temperature. Remove Component B if also stored in the freezer and bring to room
temperature.
2. Dissolve contents of one Component A vial by adding 10 mLs of Component B or 1X HBSS Buffer
plus 20 mM HEPES. Mix by vortexing (~1-2 min) until contents of vial are dissolved. It is
important that contents are completely dissolved to ensure reproducibility between
experiments.
The following procedure is designed for preparation of the Calcium 6-QF Assay Kit Loading Buffer per vial of the Explorer Kit.
1. Remove one vial each of Calcium 6-QF Assay Reagents (Components A, C) from the freezer and
equilibrate to room temperature. Remove Component B if also stored in the freezer and bring to
room temperature.
FLIPR Calcium 6 and Calcium 6-QF Assay Kits 6
2. Dissolve contents of one vial Component A by adding 10 mLs of Component B or 1X HBSS Buffer
plus 20 mM HEPES. Mix by vortexing (~1-2 min) until contents of vial are dissolved. It is
important that contents are completely dissolved to ensure reproducibility between
experiments. Transfer contents to a polypropylene tube.
3. Dissolve one vial of Component C in 25L DMSO, mix by pipetting. Transfer contents to same
tube as listed in step 2.
4. Rinse vial of Component C with 100L Component B or 1X HBSS Buffer plus 20 mM HEPES, and
transfer content to same tube as listed in step 2. Mix by vortexing (~1-2 min).
Note: If the cells require probenecid (such as CHO or other cells containing an organic anion
transporter), then a 500 mM stock solution should be prepared by adding 1 N NaOH in tissue culture
treated water, vortexing and diluting to 250 mM with 1X HBSS buffer plus 20 mM HEPES. Prepare
the Loading Buffer so that the final in-well working concentration is 2.5 mM. Adjust Loading Buffer
pH to 7.4 after addition of probenecid. Refer to the procedure for making probenecid on page four.
It may also be possible to run with less probenecid or none at all if the target is sensitive to
probenecid. Assay development is required to determine the best concentration
Do not store frozen aliquots of Loading Buffer with probenecid and always prepare fresh probenecid
on the day of the experiment. Water soluble probenecid may also be used following supplier
instructions.
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.
C. Cell Loading Using Loading Buffer
Remove cell plates from the incubator or centrifuge. For the Calcium 6 kit, it is not necessary to remove
the culture media. For the Calcium 6-QF kit it is important to remove the culture media and replace it
with HBSS + 20 mM HEPES to maintain the signal.
1. 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: Molecular Devices does not recommend washing cells before dye loading. However,
growth medium and serum may interfere with certain assays. In this case, the supernatant can
be aspirated and replaced with an equal volume of serum-free HBSS plus 20 mM HEPES buffer
before adding the Loading Buffer. Alternatively, cells can be grown in reduced serum or serum-
free conditions.
2. After adding dye, incubate cell plates for 2 hours at 37oC then keep the plates at room
temperature until used (loading time should be optimized for each cell line and target).
Note: some assays perform optimally when the plates are incubated at room temperature or
for different loading times.
Warning: Do NOT wash the cells after dye loading for either the Calcium 6 or Calcium 6-QF kits.
FLIPR Calcium 6 and Calcium 6-QF Assay Kits 7
D. Running the Calcium Mobilization Assay
FLIPR® Instrument
1. After incubation, transfer the plates directly to the FLIPR® instrument and begin the calcium
assay as described in the instrument manual.
2. When performing a signal test prior to an experiment, adjust typical average baseline counts to
range from 8,000 – 12,000 RFU (FLIPR 1, FLIPR384, or FLIPR3 instruments), 800-1,100 RFU on the
FLIPR Tetra instrument with EMCCD camera, or 5,000 – 7,000 RFU on FLIPR Tetra with ICCD
camera.
3. Suggested experimental setup parameters for each FLIPR system are listed in Tables 6, 7, and 8:
Faster addition speeds closer to the cell monolayer are recommended to ensure better mixing
of compounds and lower signal variance across the plate. However, further assay development,
adjustment of the volume, height and speed of dispense, is recommended to optimize the
individual cell response.
Table 4: Experimental setup parameters for FLIPR 1, FLIPR384, and FLIPR3 instruments
Parameters
96-well plate FLIPR 1, FLIPR384
384-well plate FLIPR384 384-well plate FLIPR3
Exposure (sec) 0.4 0.4 0.4
Camera Gain N/A N/A 50-80
Addition Volume (L) 50 12.5 12.5
Addition Height (L) 210-230 35-45 35-45
Compound Concentration (Fold) 5X 5X 5X
Addition Speed (L/sec) Adherent Cells
50-100 10-20 25-40
Addition Speed (L/sec) Non adherent Cells
10-20 5-10 10-25
Table 5: Experimental setup parameters for FLIPR Tetra system with EMCCD camera