User Manual-ENZ-51016&17
Contents
1. Enzo Enabling Discovery in Life Science FluoForte Calcium Assay Kit for microplates Instruction Manual Cat No ENZ 51016 High Throughput 100 plates kit Cat No ENZ 51017 Starter Pack 10 plates kit For research use only Rev 1 1 3 December 2010 Notice to Purchaser The FluoForte Calcium Assay Kit for microplates is a member of the CELLestial product line reagents and assay kits comprising fluorescent molecular probes that have been extensively benchmarked for live cell analysis applications CELLestial reagents and kits are optimal for use in demanding cell analysis applications involving confocal microscopy flow cytometry microplate readers and HCS HTS where consistency and reproducibility are required These products are manufactured and sold by ENZO LIFE SCIENCES INC for research use only by the end user in the research market and is not intended for diagnostic or therapeutic use Purchase does not include any right or license to use develop or otherwise exploit these product commercially Any commercial use development or exploitation of these products or development using these products without the express prior written authorization of ENZO LIFE SCIENCES INC is strictly prohibited Limited Warranty These products are offered under a limited warranty The products are guaranteed to meet appropriate specifications described in the package insert at the time of shipment Enzo Life Sciences sole obli2. ENZO LIFE SCIENCES c o Covalab s a s 13 Avenue Albert Einstein FR 69100 Villeurbanne France T 33 472 440 655 F 33 437 484 239 E info fr enzolifesciences com www enzolifesciences com ALEXIS assay designs BIOMOL NTERNATIONAL Stressgen
3. lyophilized Reagent B Dye efflux inhibitor 10x 1mL 10x 10 mL Reagent C Hanks buffer with 1 x 100 mL Not included 20 mM HEPES HHBS Additional Materials Required A fluorometric imaging plate reader capable of performing quantitative optical screening for cell based kinetic assays Molecular Devices FLIPR PerkinElmer CellLux Hamamatsu FDSS system or similar instru mentation Calibrated adjustable precision pipetters preferably with disposable plastic tips Deionized water Anhydrous DMSO Serum optional Growth medium e g Dulbecco s modified Eagle medium D MEM 10X Hanks Balanced Salt Solution HBSS e g Invitrogen 14065 056 1M HEPES Buffer e g Invitrogen 15630 080 Assay Plates 96 or 384 well black wall clear bottom plates or 1536 well low base black wall clear bottom plates 1536 well lids Compound plates 96 well or 384 well polypropylene plates 1536 well polystyrene plates Safety Warnings and Precautions This product is for research use only and is not intended for diagnostic purposes Some components of this kit may contain hazardous substances They can be harmful if ingested or absorbed through the skin and may cause irritation to the eyes The reagents of the kit should be treated as possible mutagens and should be handled with care and disposed of properly Observe good laboratory practices Gloves lab coat and protective eyewear should always be worn Never pipet by mo
4. tissue Folia Histochem Cytobiol 35 41 Zheng W et al 2004 High throughput assay technologies for ion channel drug discovery Assay Drug Dev Technol 5 543 552 Kiselyov K Shin DM Muallem S 2003 Signalling specificity in GPCR dependent Ca2 signalling Cell Signal 15 3 243 253 VIII Troubleshooting Guide Problem Potential Cause Suggestion Contributions to baseline Remove the medium before adding the High baseline fluo fluorescence by growth indicator dye to the wells Use the dye rescence medium and organic solution within 2 hours at room tem anion transport perature Make sure that the buffer used for the Untreated cells have Inconsistent DMSO negative control wells have the same calcium response concentration final concentration of DMSO as those in the test compounds Use 0 1 of BSA in all compound Agonists and antago buffer diluents Fast addition speeds nists may stick to the are recommended to ensure better Response is smaller pipette tips Experimen mixing of compounds and improved than expected tal setup parameters cell response Dye loading typically and dye loading time are takes between 30 minutes and one not optimized hour Optimizing the conditions for each cell line is recommended Well to well variation observed Fluorescence drop upon compound addition Use instrument manufacturer s recom mended dispenser and setup parame ters i e volume height
5. Fluo 4 and Calcium 4 dyes are widely used calcium ion indicators for in cell measure ment of agonist stimulated and antagonist inhibited calcium signaling in high throughput screening applications However their relatively weak fluorescence signals have limited their application in some challenging cell lines and with certain membrane receptors Enzo Life Sciences FluoForte Calcium Assay Kit for microplates provides a homogeneous fluorescence based assay for detecting intracellular calcium mobilization across a broad spectrum of biological targets Relative to other commercially available dyes FluoForte dye yields the brightest signal and largest assay window The kit provides a homogeneous mix and read no wash calcium mobilization assay The homogenous cell based assay for calcium offers fewer steps lower variability and an easier protocol for adher ent and non adherent cell lines In addition it requires neither a washing step nor exogenous addition of a quencher dye which could adversely effect receptor ligand interaction kinetics Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored at 20 C protected from light When stored properly these reagents are stable for six months from date of receipt Avoid repeated freezing and thawing Quantity Reagent ENZ 51017 ENZ 51016 10 plates kit 100 plates kit Reagent A FluoForte dye 1 vial 10 vials
6. and speed of dispensing for compound addition Incorrect dispenser and experimental setup pa rameter used Decrease the rate of addition or seed fewer cells in the wells to avoid this problem Dislodging the cells during addition Enzo Life Sciences www enzolifesciences com Enabling Discovery in Life Science NORTH SOUTH AMERICA ENZO LIFE SCIENCES INTERNATIONAL INC 5120 Butler Pike Plymouth Meeting PA 19462 1202 USA T 1 800 942 0430 610 941 0430 F 610 941 9252 E info usa enzolifesciences com SWITZERLAND amp REST OF EUROPE ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland T 41 0619268989 F 41 0 61 926 89 79 E info ch enzolifesciences com www enzolifesciences com GERMANY ENZO LIFE SCIENCES GMBH Marie Curie Strasse 8 DE 79539 L rrach Germany T 49 0 7621 5500 526 Toll Free 0800 664 9518 F 49 0 7621 5500 527 E info de enzolifesciences com www enzolifesciences com BENELUX ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium T 32 0 3 466 04 20 F 32 0 3 466 04 29 E info be enzolifesciences com www enzolifesciences com incorporating UK amp IRELAND ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 8NL UK T 08456011488 UK customers T 44 0 1392 825900 from overseas F 44 0 1392 825910 E info uk enzolifesciences com www enzolifesciences com www enzolifesciences com FRANCE
7. ate the cell plates for 1 hour at room temperature or incubate about 45 minutes at 37 C then incubate for 15 minutes at room temperature prior to assay NOTE The incubation time should be optimized for each cell line The incubation time should be limited to 1 2 hours DO NOT wash the cells after dye loading 4 Prepare the compound plates by dissolving the compound in the buffer of choice The FluoForte Calcium Assay is optimized for an agonist addition at one fifth of the final volume 5 Run the calcium flux assay by monitoring the fluorescence at Ex 490 nm Em 525 nm with a fluorometric imaging plate reader NOTE Faster addition speeds can lead to better mixing of compounds and lower signal variance across the plate Make sure to follow the recommended experimental setup parameters provided by the instrument manufacturer before reading the plate It is also important to run the signal test before the experiment Different instruments have their own intensity range Adjust the signal test intensity to the level of 10 to 15 of the maximum instrument intensity counts For example the maximum fluorescence inten sity count for FLIPR 384 is 65 000 so the instrument settings should be adjusted to have its signal test intensity around 7 000 to 10 000 VI Expected Results In a side by side comparison of FluoForte Fluo 4 Direct assay and Cal cium 4 assay CHO M1 cells were stimulated with 200 nM of ATP FluoForte yields the br
8. ay Buffer is sufficient for one plate Unused buffer may be stored at lt 20 C up to 1 month Avoid exposure to light and repeated freeze thaw cycles 3 FluoForte Dye Loading Solution Add 10 uL of Reagent A Stock Solution from step B 1 to 10 mL of 1X Assay Buffer from step B 2 Mix well This working solution is stable for at least 2 hours at room temperature C PREPARATION OF FLUOFORTE DYE LOADING SOLUTION Using ENZ 51016 100 plates kit The following procedure is for preparation of FluoForte dye loading solution for use in 10 plates Before starting equilibrate Reagent A FluoForte dye and a vial of Reagent B to room temperature 1 Hanks Buffer with 20 mM HEPES HHBS Prepare 100 mL of HHBS by mixing the following 10 mL 10X Hanks Balanced Salt Solution HBSS 2 mL HEPES Buffer 88 mL Deionized water 2 1X Assay Buffer Mix well 90 mL of HHBS from step C 1 with the contents of 1 vial 10 mL of Reagent B 3 FluoForte Dye Loading Solution a Dissolve the contents of 1 vial of Reagent A in 100 uL of DMSO Mix well b Add 100 uL of Reagent A solution to 100 mL of 1X Assay Buffer from step C 2 Mix well This working solution is stable for at least 2 hours at room temperature 4 D CALCIUM MOBILIZATION ASSAY 1 Obtain prepared cell plates see section A 2 Add FluoForte Dye Loading Solution to each well 100 uL well for 96 well plates or 25 uL well for 384 well plates 3 Incub
9. e cells from the culture medium and then resuspend the cell pellets in FluoForte dye loading solution see section B Plate the cells using 1 25 x 10 to 2 5 x 10 cells per well at a plating volume of 100 uL per well for 96 well plates or 3 x 10 to 6 x 10 cells per well at a plating volume of 25 uL per well for 384 well plates Centrifuge the plates at 800 rpm for 2 minutes with brake off prior to starting the experiments Proceed to section D page 5 NOTE Each cell line should be evaluated on an individual basis to deter mine the optimal cell density for the intracellular calcium mobiliza tion assay B PREPARATION OF FLUOFORTE DYE LOADING SOLUTION UsING ENZ 51017 10 plates kit The following procedure is for preparation of FluoForte dye loading solution for use in 1 plate Before starting equilibrate Reagent A FluoForte dye a vial of Reagent B and Reagent C HBSS to room temperature 1 Reagent A Stock Solution Add 100 uL DMSO to the vial containing Reagent A Mix well NOTE 10 ul of the reconstituted Reagent A is enough for 1 plate The remaining unused reconstituted Reagent A can be aliquoted and stored at lt 20 C for at least one month if stored properly The tubes preferably amber vials should be capped tightly Avoid exposure to light and repeated freeze thaw cycles 2 1X Assay Buffer Mix well 9 mL of Reagent C HHBS with the contents of 1 vial 1 mL of Reagent B NOTE 10 mL of 1X Ass
10. gation is to replace the product to the extent of the purchase price All claims must be made to Enzo Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo CELLestial and FluoForte are trademarks of Enzo Life Sciences Inc FLIPR is a trademark of Molecular Devices CellLux is a trademark of PerkinElmer FDSS is a trademark of Hamamatsu Photonics Several of Enzo s products and product applica tions are covered by US and foreign patents and patents pending Contents k INIFOGUGHON aiia ai II Reagents Provided and Storage III Additional Materials Required IV Safety Warnings and Precautions V Methods and Procedures A CELL PREPARATION c ccssscscsssscssrecsesencssencussseuerseusrenaeens B PREPARATION OF FLUOFORTE DYE LOADING SOLUTION USING ENZ 51017 10 PLATES KIT C PREPARATION OF FLUOFORTE DYE LOADING SOLUTION USING ENZ 51016 100 PLATES KIT D CALCIUM MOBILIZATION ASSAY VI Expected Resulis ia ia VII References xxxs s sxsxsrsssazazenezionazazanenea VIII Troubleshooting Guide 11 Introduction The calcium ion is an important second messenger involved in many physio logical and signal transduction processes within cells Fluo 3
11. ightest signal and largest assay window shown in Figure 1 This facilitates measurements of challenging cell lines and receptors Dose responses for ATP in CHO M1 cells gave similar ECsofor all the assays shown in Figure 2 This demonstrates consistent pharmacology among the assays However relative fluorescence units RFU of FluoForte are much higher than Fluo 4 and Calcium 4 Overall FluoForte Calcium Assay kit provides an optimized assay method for monitoring G protein coupled receptors GPCRs and calcium channels Its ability to generate very strong signal enables researchers to perform calcium mobilization assays with a wide range of receptor and calcium channel targets FluoForte RFU Calcium 4 i 2 W a 5 Ei 70 Time secs Figure 1 Comparisons of FluoForte Fluo 4 Direct and Calcium 4 detection of intracellular calcium mobilization in CHO M1 cells CHO cells were seeded overnight in 40 000 cells per 100 uL per well in a 96 well black wall clear bottom costar plate The cells were incubated with 100 uL of Life Technologies Fluo 4 Direct kit Molecular Devices Calcium 4 kit both based on manufactures protocol or Enzo s FluoForte kit ATP 20 uL well was added by FlexStation to achieve concentrations of 200 nM 0A FluoForte 4 Fluo 4 Direct Feo ATP nh ATP nh Figure 2 ATP Dose Response Curves in CHO M1 cells CHO cells were seeded overnight at 40 000 cells per 100
12. uL per well in a 96 well black wall clear bottom microplate Panel A The cells were incubated with 100 uL of Life Technologies Fluo 4 Direct kit Molecular Devices Calcium 4 kit both based upon manufacturer s protocol or Enzo s FluoForte dye Panel B The cells were incu bated with 100 uL of Enzo s FluoForte Calcium assay kit or with Life Technologies Fluo 4 NW kit based upon manufacturer s protocol ATP 20 uL well was added by FlexStation to achieve the final indicated concentrations No signifi cant difference in ECs of ATP for FluoForte Fluo 4 Direct and Calcium 4 was observed shown in panel A and also comparable EC5 values were observed between FluoForte and Fluo 4 NW shown in panel B In all cases FluoForte generated the highest intensity signal Vil References 1 Martin VV Beierlein M Morgan JL Rothe A Gee KR 2004 Novel Fluo 4 analogs for fluorescent calcium measurements Cell Calcium 36 509 do Ceu Monteiro M Sansonetty F Goncalves MJ O Connor JE 1999 Flow cytometric kinetic assay ofcalcium mobilization in whole blood platelets using Fluo 3 and CD41 Cytometry 35 302 Perez Terzic C Stehno Bittel L Clapham DE 1997 Nucleoplasmic and cytoplasmic differences in thefluorescence properties of the calcium indicator Fluo 3 Cell Calcium 21 275 Tretyn A Kado RT Kendrick RE 1997 Loading and localization of Fluo 3 and Fluo 3 AM calcium indicators in sinapis alba root
13. uth Do not eat drink or smoke in the laboratory areas All blood components and biological materials should be treated as potentially hazardous and handled as such They should be disposed of in accordance with established safety procedures To avoid photobleaching perform all manipulations in low light environments or protected from light by other means V Methods and Procedures Brief Summary of Assay Work Flow Prepare cells Remove medium Add FluoForte dye loading solution Incubate plate for 1 hour Add test agents Read fluorescence NOTE PLEASE READ THE ENTIRE PROCEDURE BEFORE STARTING Allow all reagents to be used to warm to room temperature before proceeding Upon thawing of solutions gently hand mix or vortex the reagents prior to use to ensure a homogenous solution A CELL PREPARATION le Adherent Cells The day before the experiment plate the cells overnight in growth medium using 4 x 10 to 8 x 10 cells per well at a plating volume of 100 uL per well for 96 well plates or using 1 x 10 to 2 x 10 cells per well at a plating volume of 25 uL per well for a 384 well plates After overnight incubation remove the growth medium from the cell plates Then proceed to section D page 5 NOTE Itis important to remove the growth medium in order to minimize background fluorescence and compound interference with se rum or culture media 2 Non adherent Cells On the day of the experiment centrifuge th
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