flipr® calcium 6-qf assay kit about the flipr® calcium 6...

FLIPR Calcium 6 and Calcium 6-QF Assay Kits 1

FLIPR® Calcium 6 Assay Kit

Product #

R8190 (Explorer Kit)

R8191 (Bulk Kit)

R8195 (Express Kit)

FLIPR® Calcium 6-QF Assay Kit Product # R8192 (Explorer Kit)

R8193 (Bulk Kit)

R8196 (Express Kit)

About the FLIPR® Calcium 6 and 6-QF Assay Kits The FLIPR® Calcium 6 Assay Kits from Molecular Devices, LLC (hereafter referred to as Molecular Devices) provide a fast, simple, and reliable fluorescence-based assay for detecting changes in intracellular calcium. The 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 5 Cell handling 5 Preparation of Loading Buffer 6-7 Cell loading 7 Running the calcium mobilization assay 8 FLIPR® instrument settings 8 FLIPR® Tetra instrument settings (EMCCD and ICCD cameras) 9 FlexStation® instrument settings 10

EXAMPLE DATA 11-12

TROUBLE SHOOTING GUIDE 12-13

PRODUCT USE LIMITATIONS AND WARRANTY 14


INTRODUCTION

About the FLIPR® Calcium 6 and Calcium 6-QF Assay Kits

Calcium assays from Molecular Devices employ sensitive calcium indicators and masking dyes. The

Calcium 6 Assay Kits contain a new dye formulation that further enhances the calcium flux assay with an

increased signal window. Kit components are mixed with buffer and incubated for approximately two

hours with cells. During incubation, the indicator passes through the cell membrane and esterases in the

cytoplasm cleave the AM portion of the molecule. After incubation with the dye, the cells are ready to

be assayed. Once the target is activated, direct measurement of intracellular fluorescence change due to

increased calcium concentration is enabled. The masking dye, in the Calcium 6 formulation, does not

enter the cell, but significantly reduces background originating from residual extracellular fluorescence

of calcium indicator, media and other components. The Calcium 6-QF formulation is a new flexible

option for quench sensitive targets or multiplexing applications.

Some cell lines have an anion-exchange protein that requires the use of an anion reuptake inhibitor such

as probenecid to retain the commonly used calcium indicators such as FLuo-3 and Fluo-4. However the

new dye formulation for the Calcium 6 kits is more resistant to such organic anion transporters, and thus

less or no probenecid may be required for assays performed with Calcium 6 kits. This is especially useful

for evaluating targets that may be sensitive to probenecid, as well as for screening agonists and

antagonists.

Assay Principle

Figure 1. Calcium assay principle


APPLICATION The FLIPR® Calcium 6 Assay uses a newly improved calcium dye formula that further enhances the signal

window of the assay and makes difficult assays more amenable for high-throughput screening. The

Calcium 6 kit provides a homogeneous assay designed to work for the majority of GPCRs, including

Chemokine and other difficult receptors, sticky compounds and allosteric modulators, as well as with

calcium channels. In addition, the new Calcium 6-QF formulation is a flexible option for quench sensitive

targets or multiplexing applications.

Which FLIPR® Calcium 6 Assay Kit is right for your application?

Media containing serum

Assay Buffer Multiplexing Probenecid

Sensitive Cell Lines

Calcium 6 Assay Kit X X X

Calcium 6-QF Assay Kit X X X

MATERIALS AND EQUIPMENT

Kit Components

Table 1: FLIPR® Calcium 6 Assay Kit and FLIPR® Calcium 6-QF Assay Kit Contents

Reagent Description R8190 FLIPR Calcium 6 (Explorer Kit)

10 vials Component A

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 ten 96-, 384-, or 1536-well plates. Each vial is sufficient for one 96-, 384-, or 1536-well plate.

R8191 FLIPR Calcium 6 (Bulk Kit) 10 vials Component A

The entire kit is sufficient for one hundred 96-, 384-, or one hundred fifty 1536-well plates. Each vial is sufficient for assaying ten 96-, 384-, or fifteen 1536-well plates.

R8195 FLIPR Calcium 6 (Express Kit) 2 vials Component A

The entire kit is sufficient for one hundred 96-, 384-, or one hundred fifty 1536-well plates. Each vial is sufficient for assaying fifty 96-, or 384-well, seventy five 1536-well plates

R8192 FLIPR Calcium 6-QF (Explorer Kit) 10 vials Component A

1 bottle Component B 1X Hank’s Balanced Salt solution (HBSS) plus 20 mM HEPES buffer, pH 7.4

10 vials Component C

The entire kit is sufficient for ten 96-, 384-, or 1536-well plates. Each vial is sufficient for one 96-, 384-, or 1536-well plate.

R8193 FLIPR Calcium 6-QF (Bulk Kit) 10 vials Component A

10 vials Component C

The entire kit is sufficient for one hundred 96-, 384-, or one hundred fifty 1536-well plates. Each vial is sufficient for assaying ten 96-, 384-, or fifteen 1536-well plates.


R8196 FLIPR Calcium 6-QF (Express Kit) 2 vials Component A

2 vials Component C

The entire kit is sufficient for one hundred 96-, 384-, or one hundred fifty 1536-well plates. Each vial is sufficient for assaying fifty 96-, 384-, or seventy five 1536-well plates.

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)

DMSO For dissolving Component C (# D8418, Sigma or equivalent)

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.

1536-well low-base black wall, clear bottom assay plates compatible with the 1536-well pipettor head

Example: Greiner 783092 or equivalent

Storage and Handling

On receipt of the FLIPR® Calcium 6 Assay Kit or the FLIPR® Calcium 6-QF Assay 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 technology 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

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 per-well seeding densities

Cell Type (cells/well)

96-well plate

(100 L growth medium) 384-well plate

(25 L growth medium) 1536-well plate

(4 L growth medium) Adherent cells 20,000 – 80,000 5,000 – 20,000 1,500 – 5,000

Non-adherent cells 40,000 – 200,000 10,000 – 50,000 3,000 – 10,000

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. Additional volumes for the Bulk and Express Assay kits are included in Table 4.

1. Remove one vial of Calcium 6 Assay Reagent (Component A) from the freezer and equilibrate to room temperature. Also equilibrate Component B 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.

Table 4: Required volumes to formulate FLIPR 6 Calcium Assay Kits

FLIPR Calcium 6 Assay Kit

Plate Format

HBSS + 20 mM HEPES or Component B Explorer Kit

# R8190 Bulk Kit # R8191

Express Kit # R8195

96- or 384-well

Volume to dissolve Component A 10 mL 10 mL 20 mL

Additional required to bring up to volume None 90 mL 480 mL

1536-well Volume to dissolve Component A 6.5 mL 10 mL 25mL


The following procedure is designed for preparation of the Calcium 6-QF Assay Kit Loading Buffer per vial of the Explorer Kit. Additional volumes for the Bulk and Express Assay kits are included in Table 5.

1. Remove one vial each of Calcium 6-QF Assay Reagents (Components A, C) from the freezer and equilibrate to room temperature. Also equilibrate Component B to room temperature.

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, and transfer content to same tube as listed in step 2. Mix by vortexing (~1-2 min).

Table 5: Required volumes to formulate FLIPR 6-QF Calcium Assay Kits

FLIPR Calcium 6-QF Assay Kit

Plate Format HBSS + 20 mM HEPES or Component B

Explorer Kit # R8192

Bulk Kit # R8193

Express Kit # R8196

96- or 384-well

Volume to dissolve Component A 10 mL 10 mL 20 mL


Volume DMSO to dissolve Component C 25 L 250 L 1.25 mL

1536-well Volume to dissolve Component A 6.5 mL 10 mL 25 mL


Volume DMSO to dissolve Component C 25 L 250 L 1.25 mL


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

1. 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. 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: For 1536-well plates, add 2 L dye per well by using an appropriate liquid handling device

for cells.

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.

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.


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 6: 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 7: Experimental setup parameters for FLIPR Tetra system with EMCCD camera

Parameters 96-well plate 384-well plate 1536-well plate

Exposure (sec) 0.4 0.4 0.4

Camera Gain 50-130 50-130 50-130

Addition Volume (L) 50 12.5 1


Excitation LED (nm) 470-495 470-495 470-495

Emission Filter (nm) 515-575 515-575 515-575

LED Intensity (%) 80 80 80

Addition Height (L) 210-230 35-45 2

Tip Up Speed (mm/sec) 10 10 5


Addition Speed (L/sec) Non Adherent Cells 10-20 10-20 1-5


Table 8: Experimental setup parameters for FLIPR Tetra system with ICCD camera

FlexStation® Instrument

1. Recommended experimental setup parameters for the FlexStation® instrument follow: Setup

up your FlexStation instrument using a SoftMax® Pro software protocol before you read the

plate. The experimental parameters are listed in Table 9.

Table 9. Experimental setup parameters for FlexStation® instrument

Fluorescence Parameters 96-well 384-well Excitation Wavelength (nm) 485 485

Emission Wavelength (nm) 525 525

Automatic Emission Cut-Off (nm) 515 515

Other Parameters 96-well 384-well PMT Sensitivity 6 6

Pipette Height (L) 230 50

Transfer Volume (L) 50 12.5

Compound Concentration (Fold) 5X 5X

Addition Speed (Rate) Adherent Cells 3 2-3

Addition Speed (Rate) Non-Adherent Cells 1 1

2. After incubation (see notes in FLIPR® instrument assay section), transfer the assay plate directly

to the FlexStation® instrument assay plate carriage and run the assay.

3. In an individual well or column of wells, the calcium flux peak(s) should be complete within 1 to

3 minutes after addition. For an entire plate however, the protocol will not complete until all

chosen columns are finished. The assays are run one column at a time.

Parameters 96-well plate 384-well plate 1536-well plate

Exposure (sec) 0.53 0.53 0.53

Camera Gain Fixed at 2,000 Fixed at 2,000 Fixed at 2,000

Camera Gate 6% 6% 6%

Addition Volume (L) 50 12.5 1


Excitation LED (nm) 470-495 470-495 470-495

Emission Filter (nm) 515-575 515-575 515-575

LED Intensity (%) 50 50 50

Addition Height (L) 210-230 35-45 2

Tip Up Speed (mm/sec) 10 10 5


Addition Speed (L/sec) Non Adherent Cells 10-20 10-20 1-5


4. In an individual well or column of wells, the calcium flux peak(s) should be complete within 1 to

3 minutes after addition. For an entire plate however, the protocol will not complete until all

chosen columns are finished. The assays are run one column at a time.

5. It is strongly recommended that parameters be optimized for each cell line and target to deliver

the best performance for your assay.

6. Analyze the data using SoftMax® Pro software.


Data Analysis: FLIPR® Calcium 6 and Calcium 6-QF Assay Examples

Figure 2. Calcium 6 Assay Kit in media:

CHOM1 Cells: Agonism by Carbachol

-5 -4 -3 -2 -1 0 10

1

2

3

4

5

EC50 = 0.013

Z @ EC80 = 0.79

Log [CarbacholM

F

/F (

max-m

in)

CHOM1 Cells: Atropine antagonism

-5 -4 -3 -2 -1 0 10

1

2

3

4

5

IC50 = 0.004

Z @ EC80 = 0.75

Log [Atropine] M

F

/F (

max-m

in)

2a 2b Figure 2. Carbachol concentration response curve in WT3 CHO M1 cells. Cells were seeded overnight at 25 L per well in a 384-well black wall clear bottom plate. On the day of the assay, cells were incubated in media with 25

L of Calcium 6 Kit. All plates were incubated for 2 hours @ 37oC and 5% CO2. In Figure 2a, a volume of 12.5 L 5X

Carbachol was added per well as agonist during detection on a FLIPR® Tetra instrument with ICCD camera. The Z factor @ EC80 was 0.79 and the EC50 values were comparable to published values. Figure 2b shows the antagonism response to 50 nM Carbachol by Atropine. The Z factor @ IC50 was 0.75. The IC50 value is comparable to published values.

Figure 3. FLIPR Calcium 6-QF Assay Kit in Buffer:

HEK 293 Muscarinic M3 receptorcarbachol agonism

-4 -3 -2 -1 0 1 20

1

2

3

4

EC50 = 1.26

Z @ EC80 = 0.83

Log [Carbachol]M

F

/F (

max-m

in)

HEK 293 muscarinic M3 receptor:atropine antagonism

-5 -4 -3 -2 -1 0 10

1

2

3

4

IC50 = 0.001

Z @ IC50 = 0.65

Log [Atropine] M

F

/F (

max-m

in)

3a 3b

Figure 3. HEK-293 cells were seeded overnight at 25 L per well in a 384-well black wall clear bottom Poly-D-

Lysine coated plate. On the day of the assay, culture media was removed and the cells were incubated in 25 L

HBSS + 20mM HEPES and 25 L of Calcium 6-QF dye. Cells were incubated for 2 hours @ 37oC and 5% CO2. Figure

3a shows the agonist response of the endogenous Muscarinic M3 receptor to Carbachol. The EC50 value was comparable to other assays. The Z factor at EC80 was 0.83. In Figure 3b, a 5X volume of the antagonist, atropine, was added per well and the plate was incubated for 10 minutes at room temperature. A 6X concentration of

Carbachol (0.6 M final in well) was added as challenge agonist during detection on a FLIPR® Tetra instrument with ICCD camera to achieve the final indicated concentration.


Figure 4. FLIPR Calcium 6 Assay probenecid signal window comparison

CHOM1 Cells: Carbachol agonism

-5 -4 -3 -2 -1 0 10

1

2

3

4

5

Ca6 Kit -PBX

Ca6 Kit +PBX

Calcium 6

+PBX

Calcium 6

- PBX

EC50 (M) 0.014 0.019

Z @ EC80 0.79 0.68

Log [CarbacholM

F

/F (

max-m

in)

Detail graphCHOM1 Cells: Carbachol agonism assay

without probenecid

-5 -4 -3 -2 -1 0 10.0

0.5

1.0

1.5

2.0

EC50 = 0.019 M

Z @ EC80 = 0.68

Log [CarbacholM

F

/F (

max-m

in)

4a 4b Figure 4. Carbachol concentration response curve as seen in Figure 2a with additional comparison to an assay run at the same time in Calcium 6 kit dye without probenecid in the loading buffer. In figure 4a, both curves are shown. The EC50 values are both within the range of published values and very close to each other. Figure 4b is a larger version of the same curve from the assay run without probenecid. Despite the smaller signal window, the EC50 value is conserved and the Z factor @ EC80 is 0.68. This suggests that the new dye formulation is less sensitive to the organic-anion transporter and requires less, or even no, anion transporter inhibitors (probenecid) to be present in the assay system. A target that is sensitive to the presence of probenecid in the loading buffer will benefit from the ability to remove it

Troubleshooting Guide

Fluorescence drop upon compound addition (“dip”)

This may be the result of dislodging cells from the well bottom during addition. Lowering the

addition/dispense speed or adjusting addition height or both should solve the problem in this case.

Another potential reason is the dilution of the non-fluorescent compound into a plate with media

containing fluorescent components (like DMEM media). This Calcium 6 kit mediates this issue compared

to earlier developed Calcium Kits. Adding volumes greater than recommended may increase the initial

fluorescence drop. In these cases it may be necessary to adjust the volumes of the components. The

recommended volume of the Loading Buffer is 100 L for 96-well plates, 25 L for 384-well plates and 2

L for 1536-well plates.


Warning: Decreasing the final in-well concentration of the Loading Buffer may decrease the response of

the assay. If only one addition is required, then adding a higher concentration of compound in low

volume could help reduce any fluorescence drop upon addition.

Serum-sensitive cells or targets

Some cells are serum-sensitive resulting in oscillations of intracellular calcium that could interfere with

results. Also, some target receptors or test compounds may interact with serum factors. In these cases,

serum-containing growth medium should be removed prior to addition of loading buffer. The volume of

growth medium removed should be replaced with an equal volume of 1X HBSS plus 20mM HEPES buffer

before loading. Alternatively cells could be incubated overnight in lower concentrations of FBS and not

washed prior to the addition of Dye Loading Buffer.

Cells tested with buffer plus DMSO show a calcium response.

Buffer used for the negative control wells should contain the same final concentration of DMSO as is

present in the wells containing the test compounds. However, this concentration of DMSO could cause

a calcium flux. In these cases, add DMSO to the Loading Buffer such that the final concentration of

DMSO in the wells does not change after buffer addition.

Precipitation in the Reagent Buffer.

The FLIPR® Calcium 6 Assay Kits are compatible with numerous buffers. Use buffers shown to work in

previously established assays, if available.

Response is smaller than expected.

Agonists and antagonists may stick to the tips and trays. Use 0.1% BSA in all compound buffer diluents

and presoak tips in compound buffer containing 0.1% BSA. (Note: Do not use the same compound plate

for presoaking and compound addition when using a 384 Pipettor head in the FLIPR® System. Instead,

use a ‘Boat’ for the presoak.)

Apparent well-to-well variation is observed.

A liquid dispenser compatible with cell handling is recommended for use with all additions off the FLIPR®

or FlexStation® instrument if apparent well-to-well variation is observed. 1536-well plates can be loaded

in quadrants by a 384-well pipettor or 1536-well liquid handling device. In some cases spinning the

compound plate or allowing the cell plates to stand at room temperature prior to use in the assay may

decrease well-to-well variation.


Product Use Limitations and Warranty

All Molecular Devices, LLC reagent products are sold for research use only and are not intended for use

in diagnostic procedures. Reagents may contain chemicals that are harmful. Due care should be

exercised to prevent direct human contact with the reagent. The MSDS is available on the

MolecularDevices.com website for more information.

Each product is shipped with documentation stating specifications and other technical information.

Molecular Devices, LLC products are warranted to meet or exceed the state specifications. The sole

obligation of Molecular Devices, LLC and the customer’s sole remedy are limited to replacement of the

products free of charge in the event that the products fails to perform as warranted.

Molecular Devices, LLC makes no other warranties, either expressed or implied, including without

limitation the implied warranties of merchantability and fitness for a particular purpose or use.

Regional Offices USA & Canada +1-800-635-5577 • Brazil +55-11-3626-6607 • China (Beijing) +86-10-6410-8669 • China (Shanghai) +86-21-3372-1088 • Germany 00800-665-32860 • Japan (Osaka) +81-6-7174-8831 • Japan (Tokyo) +81-3-6362-5260 • South Korea +82-2-3471-9531 • UK+44-118-944-8000 Check our web site for a current listing of our worldwide distributors. www.moleculardevices.com FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. The trademarks mentioned herein are the property of Molecular Devices, LLC or their respective owners. ©2013 Molecular Devices, LLC Printed in U.S.A. 5024052 Rev. C 01/31/2013 D5042644

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