FLIPR Calcium Assay Kit
Contents
1. days without loss of activity Calcium Assay Kit Experimental Protocol A Cell Handling The FLIPR Calcium Assay Kit is designed to work with many cell types both adherent and non adherent 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 In this section we provide guidelines on how to prepare the 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 COs 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 Table 3 Suggested plating volumes and seeding densities Cell Type 96 well plate 384 well plate 1536 well plate cells well 100 uL growth 25 uL growth 4 uL growth medium medium medium Adherent cells 20 000 80 000 5 000 30 000 1 500 5 000 Non adherent cells 40 000 200 000 10 000 60 000 3 000 10 000 For non adherent cells we recommend centrifuging cells from culture medium and re suspending the pellet in culture medium on the day of the experiment It is recommended after the cells are plated to centrifuge the plates at 100 x g for up to 4 minutes with brake off Alternatively non adherent cel2. dye loading growth medium and serum may interfere with certain assays or cause a fluorescence drop upon compound addition due to media autofluorescence In this case the supernatant can be aspirated and replaced with an equal volume of serum free HBSS buffer before adding the Loading Buffer 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 loading time should be optimized for your cell line Note Some assays perform optimally when the plates are incubated at room temperature Warning Do NOT wash the cells after dye loading D Running the Calcium Mobilization Assay FLIPR 1 After incubation transfer the plates directly to FLIPR and begin the calcium assay as described in the FLIPR system manual 2 When performing a signal test prior to an experiment typical average baseline counts range BATEIA S 000 10 000 RFU FLIPR mm FLIPR y or FLIPR m or 700 1 000 RFU on FLIPR 3 Suggested experimental setup parameters for each FLIPR system are as follows Fast addition speeds close 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 your cell response FLIPR Calcium Assay Kit co Molecular Devices Table 6 Experimental setup
3. is sufficient for assaying ten 96 384 well or fifteen 1536 well plates Materials Required but not provided Table 2 Reagents and supplies Item Suggested Vendor NaOH and HCI to adjust buffer pH applies only to the bulk kit e Sigma or other chemical suppliers Probenecid inhibitor for the anion exchange protein may be required with some cell lines Prepare a stock solution of 500 mM in 1N NaOH then dilute to 250 mM in HBSS buffer Adjust pH of HBSS buffer to pH 7 4 Prepare loading buffer such that the final in well concentration of probenecid is 2 2 5 mM e Sigma P8761 or other chemical suppliers Assay plates e 96 or 384 well black wall clear bottom plates assay plates OR e 1536 well low base black wall clear bottom plates assay plates e 1536 well lids Costar Nunc BD or Greiner Greiner 783092 or E amp K Scientific EK16092 Greiner 656191 or E amp K Scientific EK26191 Compound plates e 96 or 384 well polypropylene plates e 1536 well polystyrene plates Costar Nunc BD or Greiner Costar Nunc BD or Greiner FLIPR Calcium Assay Kit T Molecular Devices Storage and Handling On receipt of the FLIPR Calcium Assay Kit store contents 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
4. the Loading Buffer by diluting the Component A vial mixture with an additional volume of 1X HBSS Buffer as outlined in Table 5 Multiple washes of the vial are necessary to completely transfer the contents Table 5 Quantities of 1X HBSS necessary to perform second dilution of Component A Plate Format Explorer Kit R8041 Bulk Kit R8033 96 or 384 well N A 90 mL 1536 well N A 55 mL Note lf your cells require probenecid then a stock solution should be prepared in 1 N NaOH and added fresh to 1X HBSS buffer adjust pH to 7 4 after addition of probenecid for preparation of the Loading Buffer so that the final in well working concentration will be 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 FLIPR Calcium Assay Kit T Molecular Devices C Loading 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 uL per well for 96 well plates 25 uL for 384 well plate Note Add 2 uL per well for 1536 well plate by using an AquaMax DW4 or equivalent device Note Although Molecular Devices does not recommend washing cells before
5. Off nm 515 515 FLIPR Calcium Assay Kit T Molecular Devices Adherent Cells Parameters 96 well 384 well PMT Sensitivity 6 6 Pipette Height uL 230 50 Transfer Volume uL 50 12 5 Compound Concentration Fold 5X 5X Addition Speed Rate 2 2 3 Addition Speed Rate Non Adherent Cells 1 1 2 After incubation see notes in FLIPR section transfer the assay plate directly to the FlexStation assay plate carriage and run the assay 3 The calcium flux peak should be complete within 1 to 3 min after addition For an entire plate however the plate will not be complete until all chosen columns are finished We recommend collecting data for a minimum of 6 min during assay development for a single column to determine appropriate assay time prior to running the entire plate 4 Analyze the data using SOFTmax Pro FLIPR Calcium Assay Kit T Molecular Devices Trouble shooting Guide FLIPR Calcium Assay Kit Fluorescence drop upon compound addition 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 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 uL for 96 well pla
6. T Molecular Devices FLIPR Calcium Assay Kit Product R8041 Explorer Kit Introduction Applications R8033 Bulk Kit About the Calcium Assay Kit The FLIPR Calcium 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 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 dislodged or 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 e Incomplete washing resulting in a significant drop in signal upon addition of test compound Molecular Devices has developed the FLIPR Cal
7. ation vs Mean 52 035 2 027 12 599 896 813 0 999 Figure 2 Carbachol dose response in CHO cells stably transfected with Muscarinic receptor 1 CHO M1 Cells were seeded overnight in 4 uL per well 2000 cells per well in a 1536 well Greiner low base plate Cells were incubated with 2 uL of the Calcium Assay Kit for 1 hour at 37 C Carbachol was added 1 0 uL well to achieve the final indicated concentration n 24 FLIPR Calcium Assay Kit 10 T Molecular Devices Appendix A AquaMax DW4 protocols for dispensing into and aspirating from 1536 well plates The Molecular Devices AquaMax DW4 DW4 is the preferred device for dispensing cells and bulk reagents in 1536 well formats In addition the DW4 can remove media prior to cell loading an alternative that may be useful in situations where media or serum contributes to interferences in measuring calcium responses Here are procedures for cleaning the DW4 for using it to dispense cells and reagents and to aspirate off media Please refer to your DW4 Operator s manual for instructions on operating the washer DW4 cleaning procedure before and after dispensing cells To maintain sterility and to prevent clogging of the DW4 we recommend the following procedure before and after each daily use 1 Clean dispense head s with 10 bleach i e 1 10 of standard 6 15 sodium hypochlorite bleach 15 times Clean dispense heads with DI water 15 30 times Dry dispense head s w
8. cium Assay Kit to eliminate the causes of variability in the data as well as reducing the number of steps in the conventional wash protocol This kit offers many advantages over standard procedures for high throughput screening HTS applications Advantages of the FLIPR Calcium Assay Kit include Improvement of data quality and reproducibility 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 The FLIPR Calcium Assay Kit provides a homogeneous assay for calcium flux It is designed to work for the majority of GPCRs and calcium channels FLIPR Calcium Assay Kit T Molecular Devices Materials Kit Components Table 1 The FLIPR Calcium Assay Kit P N R8041 R8033 contents Reagent Description R8041 Explorer Kit e 10 vials Component A e 1 bottle Component B e 1X Hank s Balanced Salt solution e pH 7 4 20 mM Hepes buffer e The entire kit is sufficient for ten 96 384 or 1536 well plates Each vial is sufficient for assaying one 96 384 or 1536 well plate R8033 Bulk Kit e 10 vials Component A e 1 bottle Component B e The entire kit is sufficient for one hundred 96 384 or one hundred fifty 1536 well plates Each vial
9. ired reservoir with cells allowing 3 10 mL extra for priming Be sure 1536 heads are attached to the DW4 Select Prime on the DW4 front panel and choose the desired reservoir Prime one time Select the desired dispense protocol on the DW4 panel see section A for volumes Insert a dummy plate and dispense 2 4 rows to make sure that all inlets are dispensing Insert the assay plate and press start Place the cell plates into the hood for about 30 minutes to decrease the edge effects Repeat steps 8 and 9 for each plate Place plates in incubator or desired location Clean the DW4 as described above Protocol for adding reagents to cells This can be loading buffer as in section B 4 of the Protocol or assay buffer 1 Oar N 8 9 10 11 12 FLIPR Calcium Assay Kit Clean the DW4 as described above Prepare loading buffer as described in step B 4 of the Protocol For 1536 well plates the buffer volume is 65 mL bulk kit R8033 per vial and if required probenecid is added so that the final concentration in the well is 2 5 mM Rinse reservoir bottle with sterile PBS before filling with loading buffer Fill the desired reservoir with loading buffer allowing 3 10 mL extra for priming Be sure 1536 heads are attached to the DW4 Select Prime on the DW4 front panel and choose the desired reservoir Prime one time Select the desired dispense protocol on the DW4 panel see section C 1 for volumes I
10. ith air 3 times Clean reservoir bottle s 5 times with de ionized water Rinse reservoir bottle s 1 time with 70 Ethanol Sterilize reservoir bottle s Bottles can be autoclaved The lids can not be Dp WN repeatedly autoclaved but can be sterilized with 70 Ethanol Alternatively you may use the AquaMax Sterilant R8156 kit for cleaning the DW4 Setting up the 1536 well Parameters for the DW4 See the DW4 User Manual for details on how to set up a protocol The following suggested parameters are intended for use with the 1536 Greiner low base plate catalog 783092 1 Plate dimension set up Off set A1 7 87 Well to well center 2 25 Height 10 40 Well depth 8 6 Max volume 13 0 2 Dispense parameters Plate type 1536 Greiner low based Rows All Liquid Cells Volume dispensed 4 0ul Liquid factor 0 95 3 Aspirate parameters Plate type 1536 Greiner low based Rows All Velocity Medium speed T aspirate 1 seconds Probe height 2 mm FLIPR Calcium Assay Kit 11 T Molecular Devices Choosing DW4 file When you are ready to use these programs they are to be uploaded from computer to AquaMax DW4 file Choose desired file and push START Protocol for dispensing cells oe ao D 8 9 10 11 12 Clean the DW4 as described above Rinse reservoir bottle with sterile PBS before loading with cells Fill the des
11. ls can be treated like adherent cells plating the day before 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 B Preparation of Loading Buffer The following procedure is designed for preparation of the Loading Buffer per vial of the Explorer Kit R8041 the Bulk Kit R8033 FLIPR Calcium Assay Kit Molecular Devices 1 Explorer Kit R8041 ready to use 1X HBSS Reagent Buffer is included with this kit Bulk Kit R8033 To prepare 1X HBSS Reagent Buffer pipette 10 mL of 10X Reagent Buffer pH 5 98 Component B and dilute to 100 mL with distilled water Adjust to pH 7 4 with NaOH Note Occasionally a white precipitate will form in the 10X Reagent Buffer bottle This is normal and will re dissolve when the reagent is mixed at room temperature 2 Remove one vial of FLIPR Calcium Assay Reagent Component A and equilibrate to room temperature 3 Dissolve contents of Component A vial by adding the appropriate amount of 1X HBSS Reagent Buffer as outlined in Table 4 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 Quantities of 1X HBSS necessary to dissolve Component A contents Plate Format Explorer Kit R8041 Bulk Kit R8033 96 or 384 well 10 mL 10 mL 1536 well 6 5 mL 10 mL 4 Prepare
12. nsert a dummy plate and dispense 2 4 rows to make sure that all inlets are dispensing Insert the assay plate and press start Repeat steps 8 and 9 for each plate Place plates in incubator or desired location Clean the DW4 as described above 12 Molecular Devices Protocol for aspiration of media 1 Clean the DW4 as described above 2 Be sure the 1536 aspiration head is attached to the DW4 3 Select Clean on the DW4 front panel to clean the aspiration head 4 When prompted insert a liquid tray with sterile PBS in it 5 Press Start to clean the aspiration head 6 Select the desired aspirate protocol on the DW4 panel 7 Insert the assay plate and press start 8 Repeat step 7 for each plate 9 Place plates in incubator or desired location 10 Clean aspiration head with water a solution of 10 bleach then water 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 war
13. parameters for FLIPR FLIPR amp FLIPR Parameters 96 well plate 384 well plate 384 well plate FLIPR FLIPR FLIPR FLIPR Exposure sec 0 4 0 4 0 4 Camera Gain N A N A 50 80 Addition Volume uL 50 12 5 12 5 Addition Height uL 210 230 35 45 35 45 Compound Concentration 5X 5X 5X Fold Addition Speed uL sec 50 100 10 20 25 40 Adherent Cells Addition Speed uL sec 10 20 5 10 10 25 Non adherent Cells Table 7 Experimental setup parameters for FLIPR 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 uL 50 12 5 1 Compound Concentration 5X 5X 7X Fold Excitation LED nm 470 495 470 495 470 495 Emission Filter nm 515 575 515 575 515 575 Intensity 80 80 80 Height uL 210 230 35 45 2 Addition Speed 50 100 30 40 4 7 Adherent Cells uL sec Addition Speed 10 20 10 20 1 5 Non Adherent Cells uL sec Tip Up Speed mm sec 10 10 5 FlexStation 1 Recommended experimental setup parameters for the FlexStation are as follows Set up your FlexStation using SOFTmax Pro before you read the plate Table 8 Experimental setup parameters for 96 and 384 well plates on FlexStation Fluorescence Parameters 96 well 384 well Excitation Wavelength nm 485 485 Emission Wavelength nm 525 525 Emission Cut
14. ranted 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 Molecular Devices Corporation is the exclusive licensee of a patented assay technology from Bayer A G U S patent number 6 420 183 European patent number 0 906 572 and family members throughout the world We are the only authorized provider of any assay incorporating enhancing optimizing quenching or masking agents as claimed in Bayer s patented technology The purchase of this assay kit from Molecular Devices Corporation includes a non exclusive right to practice Bayer A G U S Patent 6 420 183 European Patent 0 906 572 and foreign counterparts thereof in conjunction with and only in conjunction with the use of this assay kit CJ Molecular Devices Sales Offices O dala USA 800 635 5577 UK 44 118 944 8000 Germany 49 89 9620 2340 Japan 06 6399 8211 Sunnyvale CA 94089 USA Check our web site for a current listing of our worldwide distributors Email info moldev com www moleculardevices com FLIPR and SOFTmax are registered trademarks and FlexStation is a trademark of Molecular Devices Corporation All other trademarks are the property of their respective owners 2003 Molecular Devices Corporation Printed in U S A R3206 Rev G 2 9 06 FLIPR Calcium Assay Kit 13
15. tablished 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 AquaMax DW4 or equivalent dispenser is recommended for use with all additions off the FLIPR or FlexStation if apparent well to well variation is observed In some cases allowing the plates to stand at room temp prior to use in the assay may decrease well to well variation ka Molecular Devices Data Analysis FLIPR Assay Examples Multiple Well Overlay Mut plea Well Owerlay Figure 1 Comparison of data output from FLIPR displaying typical calcium assay signal responses in HEK293 cells stimulated with 10 uM carbachol The left graph is a standard Fluo 3 wash assay The right graph is the FLIPR Calcium Assay Kit Notice that the overall signals are higher and more consistent with the FLIPR Calcium assay kit The CV across the 96 well plate using the FLIPR Calcium Assay Kit was 1 6 FLIPR Calcium Assay Kit Molecular Devices FLIPR 4 1536 well Assay Examples RFU 0 1 1 10 100 1000 10000 Carbachol nM y A D 1 x C B D A B c D R 2 Plot 1 Ca kit 1536 Concentr
16. tes 25 uL for 384 well plates and 2 uL 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 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 Assay Kit is compatible with numerous buffers Use buffers shown to work in previously es
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