INTRODUCTION. Assay Principle 2 Applications 3. Kit Components 3 Materials required but not provided 4 Storage and handling 4

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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 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 Page TROUBLE SHOOTING GUIDE PRODUCT USE LIMITATIONS AND WARRANTY 13 FLIPR Calcium 6 and Calcium 6-QF Assay Kits 1

2 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 FLIPR Calcium 6 and Calcium 6-QF Assay Kits 2

3 APPLICATION The FLIPR Calcium 6 Evaluation Kit includes 3 explorer-size vials each of Calcium 6 and 3 vials each or Calcium 6-QF. It is designed to help you evaluate which of the FLIPR Calcium Kits will work best with your target, whether it be GPCR or calcium channel. FLIPR Calcium 6 kits use 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. Identification of the optimal kit for your applications will result in the following improvements for most cell systems: Increased assay signal; Reduced background; Increased z- factor; Ability to multiplex with luminescent assays (QF format only) Which FLIPR Calcium 6 Assay Kit is right for your application? Media containing Probenecid Assay Buffer Multiplexing serum 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 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 well plate. Materials required but not provided Table 2: Reagents and supplies Item 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. Suggested Vendor 10X Hank s Balanced Salt Solution (# , 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 3

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 -20 o 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 37 o C, 5% CO 2 Prepare the loading buffer the following day Remove cell plates from the incubator o 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) 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 37 o C, 5% CO 2 Prepare compound plates Run experiment on FLIPR or FlexStation instruments FLIPR Calcium 6 and Calcium 6-QF Assay Kits 4

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% CO 2, 37 C 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 Cells/Well Cells/Well Cell Type (cells/well) 96-well plate (100 L growth medium) 384-well plate (25 L growth medium) Adherent cells 20,000 80,000 5,000 20,000 Non-adherent cells 40, ,000 10,000 50,000 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 dyeloaded 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 5

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 25 L DMSO, mix by pipetting. Transfer contents to same tube as listed in step Rinse vial of Component C with 100 L 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 serumfree conditions. 2. After adding dye, incubate cell plates for 2 hours at 37 o C 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 6

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, FLIPR 384, or FLIPR 3 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. Parameters Table 4: Experimental setup parameters for FLIPR 1, FLIPR 384, and FLIPR 3 instruments 96-well plate FLIPR 1, 384-well plate FLIPR 384 FLIPR 384 Exposure (sec) Camera Gain N/A N/A Addition Volume ( L) Addition Height ( L) Compound Concentration (Fold) 5X 5X 5X Addition Speed ( L/sec) Adherent Cells Addition Speed ( L/sec) Non adherent Cells well plate FLIPR 3 Table 5: Experimental setup parameters for FLIPR Tetra system with EMCCD camera Parameters 96-well plate 384-well plate Exposure (sec) Camera Gain Addition Volume ( L) Compound Concentration (Fold) 5X 5X Excitation LED (nm) Emission Filter (nm) LED Intensity (%) Addition Height ( L) Tip Up Speed (mm/sec) Addition Speed ( L/sec) Adherent Cells Addition Speed ( L/sec) Non Adherent Cells FLIPR Calcium 6 and Calcium 6-QF Assay Kits 7

8 Table 6: Experimental setup parameters for FLIPR Tetra system with ICCD camera Parameters 96-well plate 384-well plate Exposure (sec) Camera Gain Fixed at 2,000 Fixed at 2,000 Camera Gate 6% 6% Addition Volume ( L) Compound Concentration (Fold) 5X 5X Excitation LED (nm) Emission Filter (nm) LED Intensity (%) Addition Height ( L) Tip Up Speed (mm/sec) Addition Speed ( L/sec) Adherent Cells Addition Speed ( L/sec) Non Adherent Cells 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 7. Table 7. Experimental setup parameters for 96- and 384-well plates on the FlexStation instrument Fluorescence Parameters 96-well 384-well Excitation Wavelength (nm) Emission Wavelength (nm) Automatic Emission Cut-Off (nm) Other Parameters 96-well 384-well PMT Sensitivity 6 6 Pipette Height ( L) Transfer Volume ( L) Compound Concentration (Fold) 5X 5X Addition Speed (Rate) Adherent Cells Addition Speed (Rate) Non-Adherent Cells 1 1 FLIPR Calcium 6 and Calcium 6-QF Assay Kits 8

9 F/F (max-min) F/F (max-min) 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. 4. It is strongly recommended that parameters be optimized for each cell line and target to deliver the best performance for your assay. 5. 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 CHOM1 Cells: Atropine antagonism 5 4 EC 50 = EC 80 = IC 50 = EC 80 = Log [Carbachol M Log [Atropine] M 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 37 o C and 5% CO 2. 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 EC 80 was 0.79 and the EC 50 values were comparable to published values. Figure 2b shows the antagonism response to 50 nm Carbachol by Atropine. The Z IC 50 was The IC 50 value is comparable to published values. FLIPR Calcium 6 and Calcium 6-QF Assay Kits 9

10 F/F (max-min) F/F (max-min) Figure 3. FLIPR Calcium 6-QF Assay Kit in Buffer: HEK 293 Muscarinic M3 receptor carbachol agonism HEK 293 muscarinic M3 receptor: atropine antagonism EC 50 = 1.26 M EC 80 = IC 50 = IC 50 = Log [Carbachol] M Log [Atropine] M 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 37 o C and 5% CO 2. Figure 3a shows the agonist response of the endogenous Muscarinic M3 receptor to Carbachol. The EC 50 value was comparable to other assays. The Z factor at EC 80 was 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. FLIPR Calcium 6 and Calcium 6-QF Assay Kits 10

11 F/F (max-min) F/F (max-min) Figure 4. FLIPR Calcium 6 Assay probenecid signal window comparison 4a CHOM1 Cells: Carbachol agonism Ca6 Kit +PBX Ca6 Kit -PBX Log [Carbachol M Calcium 6 Calcium 6 +PBX - PBX EC 50 ( M) EC b Detail graph CHOM1 Cells: Carbachol agonism assay without probenecid EC 50 = M EC 80 = Log [Carbachol M 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 EC 50 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 EC 50 value is conserved and the Z EC 80 is 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. FLIPR Calcium 6 and Calcium 6-QF Assay Kits 11

12 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 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. FLIPR Calcium 6 and Calcium 6-QF Assay Kits 12

13 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 Brazil China (Beijing) China (Shanghai) Germany Japan (Osaka) Japan (Tokyo) South Korea UK Check our web site for a current listing of our worldwide distributors. 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 Molecular Devices, LLC Printed in U.S.A Rev. C 01/31/2013 D FLIPR Calcium 6 and Calcium 6-QF Assay Kits 13