Human Apoptosis 3-Plex Panel
Contents
1. Invitrogen Human Apoptosis 3 Plex Panel For simultaneous quantitative determination of cleaved caspase 3 175 176 cytochrome c and cleaved PARP 214 215 in human cell lysates and tissue homogenates Catalog no LHO0007 Rev 1 0 15 July 2010 PRLHO0007 Table of Contents Tableof Contents cea achte selten see Bass ill Kit Contents and Stora ge cete AAN Ga a NE ere eee e aa a a A a iv IntrOdUC 110 7 auo Na Na a Ka ME A aa AN a aaa a 1 Overview eee URP Ip aa Pere i aa N Aa a a desees eoe et eene ins 1 ASU OS coit assit ee 4 Before Starting noe HE ES IBS ES SG A ROE ee 4 Preparing Reagents au nennen sense sense been eene gnat 7 Preparing Samples anni bonn Ga a eue de feed aia awi 10 Method of Washing sesana sasaka en sinne nennen einen dagean 13 General Assay Procedure eae DI eH si 15 Hire NI sas an AN Ng naa Na aa a a A aa aa a E a An E A 19 Performance Characteristics and Limitations of the Procedure 20 Troubleshooting sese ener a aana ea nana nana aa naen 21 AppendiX ii nnde iid een 24 Te hnical SUppOIt terere ar MER IR Ee RD RU Eae DER naga 24 Purchaser Notification oet rem rte eter terree cerea gene 25 R f rences od dep C ed e rod ee de Og ena tte de dep rers 26 General Protocol Summary eese essen Ka a daa teen netten nennen 27 Plate Plan Template cuoi t ih eee a t ede ER RN 28 Explanation of Symbols s 5050505 NGAEN BATANGAN2. 2005 Sustained expansion of NKT cells and antigen specific T cells after injection of a galactosyl ceramide loaded mature dendritic cells in cancer patients J Exp Med 201 1503 1517 Kinter A et al 2004 CD25 CD4 regulatory T cells from the peripheral blood of asymptomatic HIV infected individuals regulate CD4 and CD8 HIV specific T cell immune responses in vitro and are associated with favorable clinical markers of disease status J Exp Med 200 331 343 Pickering A et al 2004 Cytokine response to infection with bacillus anthracis Spores Infect Immun 72 6382 6389 Piqueras B et al 2006 Upon viral exposure myeloid and plasmacytoid dendritic cells produce 3 waves of distinct chemokines to recruit immune effectors Blood 107 6 2613 2618 Raza K et al 2005 Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin Arthritis Res amp Ther 7 4 R784 R795 Rice P et al 2005 Oral delivery and gastrointestinal absorption of soluble glucans stimulate increased resistance to infectious challenge J Pharmacol Exp Ther 314 3 1079 1086 Szodoray P et al 2004 Circulating cytokines in primary Sjorens Syndrome determined by a multiplex cytokine system Scand J Immunol 59 592 599 Talwar S et al 2006 Gene expression profiles of peripheral blood leukocytes after endotoxin challenge in humans Physiol Geno
3. Lack of a tight seal Hold the plate firmly against the vacuum manifold to form a tight seal If only a partial plate is being analyzed cover the empty wells with a self adhesive plate seal In house controls Incorrect concentration The standard proteins included in perform differently entered in data analysis Invitrogen s Singleplex Bead Kits in subsequent software are calibrated to NIBSC assays preparations whenever possible This calibration assures lot to lot consistency in performance However the concentration of the reconstituted standards may vary with each new lot of standard Therefore it is important to check the concentration of the standard listed on the Technical Data Sheet and to verify all concentration values entered into the data analysis software Improper reconstitution or Check standard reconstitution and dilution of the standard dilution as described on page 8 Continued on next page 22 Troubleshooting Continued Problem Leaky plate Cause Solution remains on the bottom of the wells after vacuum aspiration causing wicking and leakage of well contents during next incubation Filter plate membrane tearing Solution After final wash step and plate taps use a clean absorbent towel to blot the bottom of the filter plate before addition of next liquid phase or data acquisition step Excessive vacuum can cause the membrane to tear resulting in antibody bead loss Prevent any vacuum su
4. Buffer Invitrogen Cat FNN0011 e 10 mM Tris pH 7 4 e 100mM NaCl e imMEDTA e mM EGTA e 1mM NaF 20 mM Na4P 0 e 2mM Na VO e 1 Triton X 100 e 10 glycerol e 0 196 SDS e 0 596 deoxycholate Add FRESH to the Lysis Buffer just before use 1 mM PMSF stock 0 3 M in DMSO Protease inhibitor cocktail Sigma Cat no P 2714 Continued on next page 10 Preparing Samples Continued Cell Early apoptosis can be detected in cytosolic fractions using this Fractionation 3 Plex Panel A method for producing cytosolic fractions is Procedure provided below This protocol has been applied to several human cell lines Researchers should optimize the cell extraction procedures for their own application 1 Stimulate cells as desired then collect the cells by low speed centrifugation 300 x g 2 Wash the cells twice with ice cold PBS collecting the cells between washes with low speed centrifugation 300 x g 3 Dispense 1 mL of Mitochondrial Isolation Buffer formulation presented on page 12 onto the pellet Resuspend the cells in Mitochondrial Isolation Buffer by gentle mixing 4 Transfer the mixture to a 2 mL Dounce homogenizer 5 Homogenize with 6 strokes using Pestle B This step may require some optimization for individual cell or tissue types 6 Transfer the homogenate to 1 5 mL Eppendorf microfuge tubes 7 Centrifuge the homogenate 10 minutes at 600 x g Transfer the supernatant from this step to a clean
5. Luminex Corporation and sold by Invitrogen and other vendors For Research Use Only CAUTION Not for human or animal therapeutic or diagnostic use Advances in the field of cell biology have defined a complex and interdependent set of extracellular and intracellular signaling molecules that control normal cell function There is growing interest among researchers as well as drug discovery groups in simultaneously monitoring multiple components of signaling pathways Solid phase multiplex protein assays are the tools of choice in these studies as they maximize efficiency by simultaneously profiling several proteins within individual samples Invitrogen s Multiplex Bead Immunoassays are solid phase protein immunoassays that use spectrally encoded antibody conjugated beads as the solid support The spectral beads are suitable for use in singleplex assays or may be mixed for multiplex assays according to the researcher s requirements Each assay is carefully designed and tested to assure that sensitivity range and correlation are maximized The assay is performed in a 96 well plate format and analyzed with a Luminex 100 or 200 instrument which monitors the spectral properties of the capture beads while simultaneously measuring the quantity of associated fluorophore Standard curves generated with this assay system extend over several orders of magnitude of concentrations while the sensitivity and quantitation of the assays are comparable t
6. factor during sample calculations e The influence of various drugs aberrant sera hemolyzed hyperlipidemic jaundiced etc and the use of biological fluids in place of serum plasma and tissue culture supernatant samples have not been thoroughly investigated The rate of degradation of analytes in various matrices may not have been investigated The immunoassay literature contains frequent references to aberrant signals seen with some sera attributed to heterophilic antibodies Though such samples have not been seen to date the possibility of this occurrence cannot be excluded 20 Troubleshooting Introduction Refer to the table below to troubleshoot problems encountered with the use of Invitrogen s Multiplex Bead Kits on the Luminex platform To troubleshoot problems with the Luminex instrument refer to the manual supplied with the instrument For more troubleshooting solutions visit www invitrogen com luminex Problem Cause During data Bead aggregation analysis insufficient and or erratic bead count is observed Loss of beads due to the filter plate membrane tearing Clog in instrument or probe Probe height set incorrectly Solution Make sure to vortex the beads for 30 seconds and then sonicate the beads for at least 30 seconds prior to beginning the assay to break up any bead aggregates Empty wells and add fresh wash buffer Shake for 2 to 3 minutes to resuspend the beads To prev
7. kit components at 2 to 8 C when not in use Allow all reagents to warm to room temperature before use air warm all reagents at room temperature for at least 30 minutes or alternatively in a room temperature water bath for 20 minutes except plate and standard vials The fluorescent beads are light sensitive Protect the beads from light to avoid photobleaching of the embedded dye Use aluminum foil to cover test tubes used in the assay Cover filter plates containing beads with an opaque or aluminum foil wrapped plate cover Since the amber vial does not provide full protection keep the vial covered in the box or drawer when not in use Do not expose beads to organic solvents Do not place filter plates on absorbent paper towels during loading or incubations as liquid may be lost due to contact wicking An extra plate cover is a recommended surface to rest the filter plate Following plate washing remove excess liquid and blot from the bottom of the plate by pressing the plate on clean paper towels When pipetting reagents maintain a consistent order of addition from well to well to ensure equal incubation times for all wells To prevent filter tearing avoid touching the filter plate membrane with pipette tips Do not use reagents after kit expiration date It is recommended that in house controls be included with every assay If control values fall outside pre established ranges the assay may be suspect Contact Invitrogen Techni
8. 30 seconds then sonicate for at least 30 seconds immediately prior to use in the assay Pipette 25 uL of the diluted bead solution into each well Once the beads are added to the plate keep the plate protected from light Add 200 uL Working Wash Solution to the wells Allow the beads to soak for 15 to 30 seconds Aspirate the Working Wash Solution from the wells with the vacuum manifold Repeat this washing step Blot the bottom of the filter plate on clean paper towels to remove any residual liquid Note Place the filter plate on a plate cover or non absorbent surface before all incubations 9 10 11 12 To wells designated for the standard curve pipette 100 uL of appropriate standard dilution To the wells designated for the sample pipette 100 uL of sample See Cell Fractionation Procedure per proper sample preparation page 10 Suggested total protein per well 1 to 2 ug However the exact amount should be determined by the individual user Cover filter plate containing beads with an aluminum foil wrapped plate cover Incubate the plate for 2 hours at room temperature on an orbital shaker Shaking should be sufficient to keep beads suspended during the incubation 500 600 rpm Larger radius shakers will need a lower speed and smaller radius shakers will typically handle higher speeds without splashing Ten to fifteen minutes prior to the end of this incubation prepare the detector antibody and then proceed t
9. E GAGANA entere enne 29 iii Kit Contents and Storage Storage All components of the Human Apoptosis 3 Plex Panel are shipped at 2 to 8 C Upon receipt store all kit components at 2 to 8 C Do not freeze Contents The components and amounts included in the Human Apoptosis 3 Plex Panel are listed below Reagents Provided 100 Test Kit Human Apoptosis 3 Plex Antibody Bead Concentrate 10X 0 25 mL x 1 vial contains 0 05 sodium azide Human Apoptosis 3 Plex Standard contains 0 1 sodium azide 2 vials Human Apoptosis 3 Plex Detection Antibody Concentrate 10X 1 0 mL x 1 vial contains 0 196 sodium azide Wash Solution Concentrate 20X contains 0 1 sodium azide 15 mL x 1 bottle Assay Diluent contains 0 196 sodium azide 15 mL x 1 bottle RPE Diluent contains 0 1 sodium azide 12 mL x 1 bottle Apoptosis 3 Plex RPE Concentrate 10X contains 0 1 sodium 1 mL x 1 vial azide Detector Antibody Diluent contains 3 3 mM thymol 12 mL x 1 bottle 96 well Filter Plate 1 x 96 well plate iv Overview Purpose Background Information Introduction Invitrogen s Multiplex Bead Immunoassay Kits are developed to maximize flexibility in experimental design permitting the measurement of one or multiple proteins in panels designed by the researcher The Human Apoptosis 3 Plex Panel contains a set of common reagents that are intended for use with the Luminex 100 or 200 dual laser detection system manufactured by
10. ate for 2 hours Add detection antibody then incubatefor 1 hour Add streptavidin RPE then incubate for 30 minutes Resuspend and acquire data using Luminex Detection system Methods Before Starting Materials Required but Not Provided Luminex xMAP system with data acquisition and analysis software Invitrogen Cat no MAP0200 contact Invitrogen for instrument and software placement services see page 24 Filtration vacuum manifold for bead washing Pall Cat no 5017 is recommended Sonicating water bath Vortex mixer Orbital shaker small diameter rotation recommended Calibrated adjustable precision pipettes preferably with disposable plastic tips A manifold multi channel pipette is desirable Distilled or deionized water Glass or polypropylene tubes Aluminum foil or opaque 96 well plate cover Invitrogen Cat no PC10 Continued on next page Before Starting Continued Procedural Notes Review the procedural notes below before starting the protocol This kit has not been tested for multiplexing with other markers Should the user elect to multiplex this kit with other Luminex kits the assay conditions should be determined empirically for each specific application Do not invert the filter plates during the assay The filter plates are designed to be used in conjunction with a vacuum manifold do not exceed 5 mm Hg and emptied from the bottom Do not freeze any component of this kit Store
11. cal Support for product and technical assistance Do not mix or substitute reagents with those from other lots or sources Continued on next page Before Starting Continued Recommended Plate Plan Note Handle all blood components and biological materials as potentially hazardous Follow standard precautions as established by the Centers for Disease Control and Prevention and by the local Occupational Safety and Health Administration when handling and disposing of infectious agents This kit contains materials with small quantities of sodium azide Sodium azide reacts with lead and copper plumbing to form explosive metal azides Upon disposal flush drains with a large volume of water to prevent azide accumulation Avoid ingestion and contact with eyes skin and mucous membranes In case of contact rinse affected area with plenty of water Observe all federal state and local regulations for disposal It is recommended that a plate plan be designed before starting the assay A plate plan template is provided on page 28 for a fill in template The following is a suggested plate plan B blank Assay Diluent Standards 7 through 1 lowest concentration to highest The remainder of the plate is available for controls and samples which may be run as a singlet or in duplicate as desired Running all standards samples and controls in duplicate is recommended Pre
12. dard are indicated on the Technical Data Sheet e Perform standard dilutions in glass or polypropylene tubes Continued on next page Preparing Reagents Continued Reconstituting 1 To the standard vial add the suggested reconstitution volume Lyophilized Standards Preparing Standard Curve 300 uL Reconstituted Standard Std1 of Assay Diluent see Technical Data Sheet Do not vortex When mixing or reconstituting protein solutions always avoid foaming 2 Replace the vial stopper and allow the vial to stand undisturbed for 10 minutes 3 Gently swirl and invert the vial 2 to 3 times to ensure complete reconstitution and allow the vial to sit at room temperature for an additional 5 minutes The standard curve is made by serially diluting the reconstituted standard in Assay Diluent See below Do not vortex Mix by gently pipetting up and down 5 to 10 times 300 pL 300uL 300yL 300yuL 300pL NC CNET NS C N CN Blank M d M M M ov ov ov ov ov GY o o o Std2 Std3 Std4 Std5 Std6 Std7 Discard all remaining reconstituted and diluted standards after completing assay Return the Assay Diluent to the kit Continued on next page Preparing Reagents Continued Online Tool Go to http www invitrogen com luminex under Multiplex Solution Tools click Luminex Calculation Worksheet for auto calculation of all assay dilutions Preparing 1X Determine the number of wells required for the assay Antibod
13. ed Reverse Pipetting Recommendation Note 14 To reduce bubbles and loss of reagents due to residual fluid left in pipette tips use the recommended reverse pipetting technique 1 To reverse pipette set the pipette to the appropriate volume needed Note Do not reverse pipette volumes lt 20 uL 2 Press the push button slowly to the first stop and then press on past it Note the amount past the first stop will depend on the volume of liquid available to aspirate from 3 Immerse the tip into the liquid just below the meniscus Release the push button slowly and smoothly to the top resting position to aspirate the set volume of liquid 5 Place the end of the tip against the inside wall of the recipient vessel at an angle 6 Press the push button slowly and smoothly to the first stop Some liquid will remain in the tip this should not be dispensed 7 Remove the tip keeping the pipette pressed to the first stop Bring all reagents and samples to room temperature before use General Assay Procedure Analyte Capture 1 Use an adhesive plate cover to seal any unused wells This will keep the wells dry for future use Pre wet the designated assay wells by adding 200 uL of Working Wash Solution into designated wells Incubate plate 15 to 30 seconds at room temperature Aspirate the Working Wash Solution from the wells using the vacuum manifold Vortex the diluted bead solution prepared on page 9 for
14. ent membrane tearing place pipette tips on the side of the well rather than straight down onto the membrane when dispensing liquid into the wells Turn the vacuum manifold on before placing the filter plate on the top to prevent vacuum surge When evaluating a new vacuum manifold adjust the vacuum force so that 3 seconds are required to empty 0 2 mL from the wells of a plate Remove probe sonicate for 5 minutes rinse the probe and reinstall Run an unclog protocol See instrument manual Readjust the instrument probe height If it is too low it could puncture the well membrane If it is too high air could be pulled up with the liquid which may appear as bead fragments to the instrument Continued on next page 21 Troubleshooting Continued Problem Cause Solution During washing The filter plate is clogged Dislodge the clog by gently pushing steps the vacuum the pointed end of a 15 mL plastic manifold does not conical tube into the bottom of the aspirate the liquid plate under the clogged well This from wells of the procedure clears the small opening filter plate in the plastic casing Dislodge by placing the filter plate on a clean paper towel and use a gloved thumb or a 1 mL Pasteur pipette bulb to plunge the top of the clogged well To prevent filter plate clogging clarify samples by centrifugation at 1 000 x g for 10 minutes prior to analysis Some samples may also require filtration prior to analysis
15. equunu Boyeye gt Wy 28 Explanation of Symbols Symbol Description Symbol Description Catalogue Number RUO Research Use Only Batch code In vitro diagnostic medical device Use by aud Manufacturer Temperature limitation 8 es I sio x z 8 m zn m European Community authorised representative With contains Without does not contain Protect from light Consult accompanying documents Bux 1 Directs the user to consult instructions for use IFU accompanying the product Copyright Invitrogen Corporation 15 July 2010 29 Notes 6 invitrogen Corporate Headguarters en Corporation n Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country ific contact information visit our web site at www invitrogen com
16. f differing intensities Each bead is given a unique number or bead region allowing differentiation of one bead from another Beads of defined spectral properties are conjugated to protein specific capture antibodies and added along with samples including standards of known protein concentration control samples and test samples into the wells of a filter bottom microplate and where proteins bind to the capture antibodies over the course of a 2 hour incubation After washing the beads protein specific detector antibodies are added and incubated with the beads for 1 hour During this incubation the protein specific detector antibodies bind to the appropriate immobilized proteins After removal of excess detector antibodies an R Phycoerythrin RPE conjugated secondary antibody is added and allowed to incubate for 30 minutes The RPE conjugate binds to the detector antibodies associated with the immune complexes on the beads forming a four member solid phase sandwich After washing to remove unbound RPE conjugate the beads are analyzed with the Luminex detection system By monitoring the spectral properties of the beads and the amount of associated R Phycoerythrin RPE fluorescence the concentration of one or more proteins can be determined Experimental Overview Experimental Experimental outline for using the Human Apoptosis 3 Plex Outline Panel is shown below Prewet wells Add beads dard and samples then incub
17. ications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 25 References The references below demonstrate the success customers achieve when using Invitrogen Multiplex Assays For a complete list visit www invitrogen com luminex 10 11 Chang D H et al
18. microfuge tube Centrifuge a second time 10 minutes at 600 x g The pellets from these spins containing cell fragments and larger organelles such as nuclei may be discarded as desired 8 Transfer the resulting supernatant to a clean tube Centrifuge at 10 000 x g for 15 minutes The supernatant resulting from this spin is the cytosol The pellet is the mitochondrial fraction 9 The mitochondria may be lysed and analyzed with the assay if desired To lyse the mitochondria add 50 uL Cell Extraction Buffer formulation presented on page 12 to the pellet Incubate on ice for 15 minutes with occasional vortex mixing 10 Clarify the cytosolic and the lysed mitochondrial fractions by centrifugation at 18 000 x g for 5 minutes 11 Aliquot the cleared fractions into clean microfuge tubes and determine total protein concentration Cell fractions should be frozen and stored at 80 C or analyzed shortly after collection 11 Preparing Samples Continued Cell Fractionation Procedure 12 This kit is also suitable for use with whole cell lysates however the translocation of cytochrome c to the cytosol cannot usually be detected in this sample type A method for producing whole cell lysates is provided below 1 Stimulate cells as desired then collect the cells by low speed centrifugation 300 x g 2 Wash the cells twice with ice cold PBS collecting the cells between washes with low speed centrifugation 300 x g Add Cell Ext
19. mics 25 203 215 Wille Reece U et al 2004 Immunization with HIV 1 Gag protein conjugated to a TLR7 8 agonist results in the generation of HIV 1 Gag specific Th1 and CD8 T cell responses J Immunol 172 449 456 Williams D L et al 2005 Modulation of the phosphoinositide 3 kinase pathway alters innate resistance to polymicrobial sepsis J Immunol 174 7676 7683 Zacharowski K et al 2006 Toll like receptor 4 plays a crucial role in the immune adrenal response to systemic inflammatory response syndrome Proc Natl Acad Sci USA 103 16 6392 6397 For Research Use Only CAUTION Not for human or animal therapeutic or diagnostic use 26 General Protocol Summary Pre wet plate Add 25 uL 1X antibody coated beads and 200 uL Wash Solution Wash 1 x 200 uL Sample type Standard Cell Lysate Add 100 uL 8 g Add 100 uL appropriately i g i g standard diluted sample i i w 4 Shake for 2 hr at RT in the dark Wash 2 x 200 uL Add 100 uL 1X detector antibody Shake for 1 hour at RT in the dark Wash 2 x 200 uL Add 100 uL 1X anti Rabbit RPE Shake for 30 min at RT in the dark Wash 3x 200 uL Add 100 uL wash buffer Shake for 2 3 min Read in Luminex Detection System Total time 3 5 hr 3 Detector R phycoerythrin Q Protein oe A antibody IX RPE 27 Plate Plan Template aeq Jequunw 101 QI 91eld J
20. n clean paper towels to remove residual liquid Add 100 uL of prepared 1X RPE conjugate to each well and incubate the plate for 30 minutes at room temperature on an orbital shaker Shaking should be sufficient to keep the beads suspended during incubation 500 600 rpm Remove the liquid from wells by aspiration with the vacuum manifold Wash beads by adding 200 uL Working Wash Solution to the wells allow the beads to soak for 10 seconds then aspirate with the vacuum manifold Repeat this washing step 2 additional times for a total of 3 washes Blot the bottom of the filter plate on clean paper towels to remove residual liquid Add 100 uL of Working Wash Solution to each well Shake the plate on an orbital shaker 500 600 rpm for 2 to 3 minutes to resuspend the beads Note If the plate cannot be read on the day of the assay cover and store the plate in the dark overnight at 2 to 8 C for reading the following day without significant loss of fluorescent intensity Aspirate Working Wash Solution from stored plates and add 100 uL fresh Working Wash Solution Place the plate on an orbital shaker for 2 to 3 minutes at 500 600 rpm prior to analysis Uncover the plate and insert the plate into the XY platform of the Luminex 100 or 200 instrument and analyze the samples Determine the concentration of samples from the standard curve using curve fitting software It is recommended to use the five parameter algorithm with a weigh
21. o Analyte Detection Step 1 Continued on next page 15 General Assay Procedure Continued Preparing 1X Detector Antibody Analyte Detection 16 The Detector Antibody is supplied as a 10X concentrate and must be diluted prior to use To prepare a 1X Detector Antibody stock dilute 10 uL of 10X Detector Antibody in 100 uL of Detector Antibody Diluent per assay well Each well requires 100 uL of the diluted Detector Antibody See table below for examples of volumes to combine Note Dilution factor is 1 11 for extra pipetting volume Number of Wells Vol 10X Detector Vol Detector Antibody Concentrate Antibody Diluent 24 0 24 mL 2 4 mL 32 0 32 mL 32mL 40 0 40 mL 4 0 mL 48 0 48 mL 4 8 mL 56 0 56 mL 5 6mL 64 0 64 mL 6 4 mL 72 0 72 mL 7 2mL 80 0 80 mL 8 0 mL 88 0 88 mL 8 8 mL 96 0 96 mL 9 6 mL After the 2 hour capture bead incubation remove the liquid from wells by aspiration with the vacuum manifold Add 200 uL of Working Wash Solution to the wells Allow the beads to soak for 15 to 30 seconds then aspirate with the vacuum manifold Repeat this washing step Blot the bottom of the filter plate on clean paper towels to remove residual liquid Add 100 uL of prepared 1 X Detector Antibody page 16 to each well and incubate the plate for 1 hour at room temperature on an orbital shaker Shaking should be sufficient to keep the beads suspended during incubation 500 600 rpm Prepare the Luminex 100 or 200 in
22. o ELISAs Enzyme Linked Immuno Sorbent Assays Assay standards are calibrated to NIBSC National Institute for Biological Standards and Controls reference preparations when available to assure accurate and reliable results Continued on next page Overview Background Information Continued Assay Overview ex 0 ec O0 Antibody Conjugated Beads 0x3 ecc 0x3 0 lt Analyte Capture ILS pa x 0 gt x lt gt Detection Antibody u zz Analyte Detection Continued Invitrogen s Human Apoptosis 3 Plex Panel is designed for quantitative determination of caspase 3 when cleaved between amino acid residues 175 and 176 caspase 3 175 176 cytochrome c and poly ADP ribose polymerase when cleaved between amino acid residues 214 and 215 PARP 214 215 human in cell lysates and tissue homogenates This kit has not been tested for multiplexing with other markers Should user elect to multiplex this kit with other Luminex kits the assay conditions should be determined empirically for each specific application Visit the Invitrogen web site for a current listing of available Invitrogen multiplex bead immunoassays and reagents at www invitrogen com luminex The xMAP technology combines the efficiencies of multiplexing up to 100 different proteins for simultaneous analysis with reproducibility similar to ELISA The technology uses 5 6 um polystyrene beads which are internally dyed with red and infrared fluorophores o
23. paring Reagents Introduction Preparing Wash Solution Guidelines for Standard Curve Preparation Review the information in this section before starting Prepare components of the Human Apoptosis 3 Plex Panel according to instructions below Note Bring all reagents and samples to room temperature before use Upon storage at 2 to 8 C a precipitate may form in the 20X Wash Solution Concentrate If this occurs warm the 20X Wash Solution Concentrate to 37 C and mix until the precipitate is dissolved 1 Prepare a 1X Working Wash Solution for use with a 96 well plate by transferring the entire contents of the Wash Solution Concentrate bottle to a 500 mL container or equivalent and then add 285 mL of deionized water Mix well 2 The 1X Working Wash Solution is stable for up to 2 weeks when stored at 2 to 8 C Note To prepare smaller volumes of 1X Working Wash Solution mix 1 part of 20X concentrate with 19 parts of deionized water Mix well Each Kit comes with 2 complete sets of standard vials so that 2 runs on the plate can be made with freshly prepared standards Reconstitute the protein standard within 1 hour of performing the assay Additional standards are available from Invitrogen custom services e Before performing standard mixing and serial dilutions confirm reconstitution volumes on the Technical Data Sheet included in the Singleplex Bead Kit s e The concentrations of the protein components of the stan
24. raction Buffer to the cell pellet recommended cell lysate concentration is 2 to 5 mg mL 3 Incubate 15 minutes on ice with occasional vortexing Transfer the lysate to a microfuge tube and centrifuge at 18 000 x g for 10 minutes at 2 to 8 C 4 Aliquot the cleared lysate into clean microfuge tubes and determine total protein concentration Whole cell lysates should be frozen and stored at 80 C or analyzed shortly after collection Avoid multiple freeze thaw cycles of frozen samples Thaw completely mix well and clarify by centrifugation 18 000 x g for 5 minutes prior to analysis to prevent clogging of the filter plates The Mitochondrial Isolation Buffer and Cell Extraction Buffer are stable for 2 to 3 weeks at 2 to 8 C or 6 months when stored in aliquots stored at 20 C Cell fractions and whole cell lysates must be diluted at least 10 fold in Assay Diluent prior to analysis Method of Washing Method of Washing Incomplete washing adversely affects assay results Perform all wash steps with the Wash Solution supplied with the kit All phases of the assay including incubations wash steps and loading beads are performed in the filter bottom plate supplied with the kit 1 To wash beads place the filter plate on the vacuum manifold and aspirate the liquid with gentle vacuum do not exceed 5 mm Hg Excessive vacuum can cause the membrane to tear resulting in antibody bead loss Prevent any vacuum surge by opening and adj
25. re available at www invitrogen com msds Purchaser Notification Limited Use Label License No 330 Luminex Assay Product Limited Warranty By opening the packaging containing this Assay Product which contains fluorescently labeled microsphere beads authorized by Luminex Corporation or using this Assay Product in any manner you are consenting and agreeing to be bound by the following terms and conditions You are also agreeing that the following terms and conditions constitute a legally valid and binding contract that is enforceable against you If you do not agree to all of the terms and conditions set forth below you must promptly return this Assay Product for a full refund prior to using it in any manner You the customer acquire the right under Luminex Corporation s patent rights if any to use this Assay Product or any portion of this Assay Product including without limitation the microsphere beads contained herein only with Luminex Corporation s laser based fluorescent analytical test instrumentation marketed under the name Luminex Instrument Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Invitrogen warrants that all of its products will perform according to specif
26. rge by opening and adjusting the vacuum on the manifold before placing the plate on the manifold surface 23 Appendix Technical Support World Wide Visit the Invitrogen website at www invitrogen com for Web e Technical resources including manuals Technical Data Sheet quick calculation worksheet application notes MSDSs FAQs formulations citations handbooks and more e Complete Technical Support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 813 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail E mail on techsupport invitrogen com ipinfo invitrogen com eurotech invitrogen com MSDS Requests 24 Invitrogen Corporation 542 Flynn Road Camarillo CA 93012 USA Tel Toll Free 1 800 955 6288 E mail techsupport invitrogen com Material Safety Data Sheets MSDSs a
27. strument during this incubation step Refer to the Luminex Instrument Quick Reference card provided in kit Refer to the Technical Data Sheet for all bead regions and standard concentration values Ten to fifteen minutes prior to the end of the detector incubation step prepare the RPE conjugate and then proceed with Assay Reading Step 1 Continued on next page General Assay Procedure Continued Preparing RPE The RPE conjugate is supplied as a 10X concentrate and must be Conjugate diluted prior to use Protect RPE conjugate from light during handling To prepare a 1X RPE conjugate stock dilute 10 uL of 10X RPE conjugate in 100 of uL RPE Diluent per assay well Each well requires 100 uL of the diluted RPE conjugate See table below for examples of volumes to combine Note Dilution factor is 1 11 for extra pipetting volume Number of Vol 10X Vol RPE Wells RPE conjugate Concentrate Diluent 24 0 24 mL 2 4 mL 32 0 32 mL 3 2 mL 40 0 40 mL 4 0 mL 48 0 48 mL 4 8 mL 56 0 56 mL 5 6 mL 64 0 64 mL 6 4 mL 72 0 72 mL 7 2 mL 80 0 80 mL 8 0 mL 88 0 88 mL 8 8 mL 96 0 96 mL 9 6 mL Continued on next page 17 General Assay Procedure Continued Assay Reading 18 10 Remove the liquid from wells by aspiration with the vacuum manifold Add 200 uL Working Wash Solution to the wells Allow the beads to soak for 15 to 30 seconds then aspirate with the vacuum manifold Repeat this washing step Blot the bottom of the filter plate o
28. ted function 1 y depending on the software package used Instrument Setup Helpful guides for Luminex 100TMand 200 users 1 QVO ee an 7 Assign the appropriate Bead Region refer to the kit specific technical data sheet to each analyte We recommend that the user count 100 events bead region Set Minimum Events to 0 Set Sample Size to 50 pil Set Flow Rate to 60 ul minute For Invitrogen kits we recommend an initial Double Discriminator DD gate setting of 7 800 15 000 This setting may vary among instruments and must be determined by the user Collect Median RFU Note All Invitrogen Multiplex Luminex Kits are qualified at low PMT setting 19 Performance Characteristics and Limitations of the Procedure Performance Refer to analyte specific Technical Data Sheet for performance Characteristics claims Procedure Do not extrapolate the standard curve beyond the highest or Limitations lowest standard point the dose response and data collected in these regions may be non linear and should be considered inaccurate Note In some cases further dilution of the standard beyond 7 points may be possible to extend the low end of the standard curve e Dilute samples that are greater than the highest standard with Assay Diluent or appropriate matrix diluent reanalyze these samples and multiply results by the appropriate dilution factor e Samples are diluted in the assay be sure to account for this dilution
29. usting the vacuum on the manifold before placing the plate on the manifold surface Stop the vacuum pressure as soon as the wells are empty Do not attempt to pull the plate off the vacuum manifold while the vacuum is still on or filter plate damage may occur Release the vacuum prior to removing the plate If solution remains in the wells during vacuum aspiration do not detach the bottom of the 96 well filter plate In some cases minor clogs in the filter plate may be dislodged by carefully pressing the bottom of the plate under the clogged well with the pointed end of a 15 mL plastic conical tube Place the filter plate on a clean paper towel and use a gloved thumb or a 1 mL Pasteur pipette bulb to plunge the top of the clogged well Empty all clogged wells entirely before continuing the washes Note Do not attempt to repetitively pull vacuum on plates with clogged wells This can compromise the unclogged wells and bead loss may occur After all wells are empty lightly tap or press the filter plate onto clean paper towels hold the plate in the center for tapping to remove excess fluid from the bottom of the filter plate Do not invert plate Following the last aspiration and plate taps use a clean absorbent towel to blot the bottom of the filter plate before addition of next liquid phase or data acquisition step Do not leave plate on absorbent surface when adding reagents Continued on next page 13 Method of Washing Continu
30. y Beads The Antibody Bead Concentrate is supplied as a 10X concentrate and must be diluted prior to use The fluorescent beads are light sensitive Protect antibody conjugated beads from light during handling 1 Immediately before dispensing vortex the 10X Antibody Bead Concentrate for 30 seconds followed by sonication in a sonicating water bath for 30 seconds 2 Prepare 1X Antibody Bead stock by diluting 2 5 uL of 10X beads in 25 uL of Working Wash Solution page 7 per assay well Each well requires 25 uL of the diluted beads See table below for examples of volumes to combine Note Dilution factor is 1 11 for extra pipetting volume Number of Wells Vol 10X Antibody Bead Vol Working Wash Concentrate Solution 24 0 06 mL 0 6 mL 32 0 08 mL 0 8 mL 40 0 10 mL 1 0 mL 48 0 12 mL 1 2 mL 56 0 14 mL 1 4 mL 64 0 16 mL 1 6 mL 72 0 18 mL 1 8 mL 80 0 20 mL 2 0 mL 88 0 22 mL 2 2mL 96 0 24 mL 2 4 mL Preparing Samples Introduction This protocol has been applied to several human cell lines Researchers should optimize the cell tissue extraction buffers and procedures for their own applications Cell Fraction Recommended Buffer Formulations Extraction Buffer Formulations Mitochondrial Isolation Buffer e 20mM HEPES pH 7 5 250 mM sucrose 10 mM KCl e 1 5 mM MgCl e 0 5 mM EDTA e 0 5 mM EGTA 1 mM DTT Add FRESH to the Mitochondrial Isolation Buffer just before use 1 mM PMSF stock 0 3 M in DMSO Cell Extraction
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