ab65313 ADP/ATP Ratio Assay Kit (Bioluminescent) - Abcam

ab65313ADP/ATP Ratio Assay Kit(Bioluminescent)Instructions for UseFor the rapid, sensitive and accuratemeasurement of the ratio of ADP/ATP in varioussamples.This product is for research use only and is notintended for diagnostic use.

Table of Contents1. Overview 32. Protocol Summary 43. Components and Storage 54. Assay Protocol 75. Data Analysis 96. Troubleshooting 112

1. OverviewThe changes in ADP/ATP ratio have been used to differentiate thedifferent modes of cell death and viability. Increased levels of ATPand decreased levels of ADP have been recognized in proliferatingcells. In contrast, decreased levels of ATP and increased levels ofADP are recognized in apoptotic cells. The decrease in ATP andincrease in ADP are much more pronounced in necrosis thanapoptosis.Abcam’s ADP/ATP Ratio Assay Kit (Bioluminescent) utilizesbioluminescent detection of the ADP and ATP levels for a rapidscreening of apoptosis, necrosis, growth arrest, and cell proliferationsimultaneously in mammalian cells.The assay utilizes the enzyme luciferase to catalyze the formation oflight from ATP and luciferin, and the light can be measured using aluminometer or Beta Counter. ADP level is measured by itsconversion to ATP that is subsequently detected using the samereaction. The assay can be fully automatic for high throughput and ishighly sensitive (detects 100 mammalian cells/well).3

2. Protocol SummaryInduce Apoptosis in CellsPrepare Reaction Mix and Add to WellsTransfer Cells to WellsRead LuminescenceAdd ADP Converting EnzymeRead Luminescence4

3. Components and StorageA. Kit ComponentsItemQuantityNucleotide Releasing Buffer50 mLATP Monitoring Enzyme1 vialADP Converting Enzyme1 vialEnzyme Reconstitution Buffer1.2 mL* Store kit at -20°C.• The reconstituted enzymes are stable for up to 2 months at+4°C after reconstitution.• For more accurate handling, the reconstitutedADP-Converting Enzyme can be diluted 10-fold withNucleotide Releasing Buffer just before use (AssayProtocol, Step 6), and then use 10 µl of the enzyme foreach assay.NUCLEOTIDE RELEASING BUFFER: Ensure the buffer is at roomtemperature before use. The optimal temperature is 22°C. Keepenzymes on ice during the assay and protect from light as much aspossible.5

B. Additional Materials Required• Microcentrifuge• Pipettes and pipette tips• Luminometer/luminescence-capable plate reader or BetaCounter• 96 well plate• Orbital shaker6

4. Assay ProtocolNotes:a) The ADP/ATP Ratio Assay Kit (Bioluminescent) is significantlymore sensitive than other methods used for cell viability assays.As a general guide, we recommend using 1 x10 4 -1 x10 6 cells perassay.b) Avoid contamination with ATP from exogenous biologicalsources, such as bacteria or fingerprints.c) The assay can be performed using either a single tube or a whitewalled 96-well luminometer plate (100 µl/well culture volume isrecommended).1. Induce apoptosis in cells by desired method. Concurrentlyincubate a control culture without induction.2. Reaction Mix:Reconstitute ATP Monitoring Enzyme with 1.1 ml of the EnzymeReconstitution Buffer & mix gently by inversion. For each samplewell to be measured, mix 100 µl of reaction mix consisting of:ATP Monitoring Enzyme 5 µlNucleotide Releasing Buffer 95 µl7

3. Add 100 µl of the reaction mix to the appropriate wells of a 96-wellplate and read the background luminescence.For higher accuracy let the reaction mix sit at room temperature toburn off low level ATP contamination for a few hours.4. Samples:a. For suspension cells: Transfer 10 µl of the cultured cells (10 3 -10 4 ) into luminometer plate.b. For adherent cells: Remove culture medium and treat cells (10 3 -10 4 ) with 50 µl of Nucleotide Releasing Buffer for 5 minutes atroom temperature with gentle shaking. Transfer into luminometerplate.5. After 5-10 min read the sample in a luminometer or luminescencecapableplate reader (Data A).6. Reconstitute ADP Converting Enzyme with 220 µl of theNucleotide Releasing Buffer & mix gently by inversion.To measure ADP levels in the cells, read the samples from Step 4after 10 min (Data B), and then add 1 µl of ADP Converting Enzyme.Read the samples again in 5-10 min in a luminometer (Data C).**Note: The results can be analyzed using cuvette-basedluminometers or Beta Counters. When Beta Counter is used itshould be programmed in the “out of coincidence” (or Luminescencemode) for measurement. The entire assay can directly be done in a8

96-well plate**. It can also be programmed automatically usinginstrumentation with injectors (When using injector the ATPMonitoring Enzyme and the ADP Converting Enzyme can be dilutedwith the Nuclear Releasing Buffer at 1:50 for injector).** Note: The assay utilizes a “glow-type” luciferase which hasreplaced the original “flash-type” luciferase. While still sensitive tosub-picomole amounts of ATP, the glow-type reactions lasts at leastan hour without any significant decline in light output. This meansthat ATP & ADP levels are now determined by the steady-state lightoutput levels. This makes the reading of an entire 96-well (384-well)plate much more feasible.5. Data AnalysisADP/ATP Ratio is calculated as:Interpretation of Results:Data C – Data BData ACell Fate ADP Level ATP Level ADP/ATPProliferation Very Low High Very LowGrowth Arrest Low Slightly Increased LowApoptosis High Low HighNecrosis Much Higher Very Low Much Higher9

Note: The interpretation of different ratios obtained may varysignificantly according to the cell types and conditions used.However, the following criteria may be used as guidelines:a. Test gives markedly elevated ATP values with no significantincrease in ADP levels in comparison to control cells =proliferation.b. Test gives similar or slightly higher levels of ATP and with little orno change in ADP compared to control = growth arrest.c. Test gives lower levels of ATP to control but shows an increase inADP = apoptosis.d. Test gives considerable lower ATP levels than control but greatlyincreased ADP = necrosis.10

6. TroubleshootingProblem Reason SolutionAssay notworkingUnexpectedresultsUse of a differentbufferProtocol step missedPlate read atincorrect wavelengthUnsuitable microtiterplate for assayMeasured at wrongwavelengthSamples containimpeding substancesUnsuitable sampletypeSample readings areoutside linear rangeRefer to protocol and usebuffers as indicatedRe-read and follow the protocolexactlyEnsure you are usingappropriate reader and filtersettings (refer to datasheet)Fluorescence: Black plates(clear bottoms);Luminescence: White plates;Colorimetry: Clear plates.If critical, datasheet will indicatewhether to use flat- or U-shapedwellsUse appropriate reader and filtersettings described in datasheetTroubleshoot and also considerdeproteinizing samplesUse recommended samplestypes as listed on the datasheetConcentrate/ dilute samples tobe in linear range11

SampleswithinconsistentreadingsLower/Higherreadings insamplesandstandardsUnsuitable sampletypeSamples prepared inthe wrong bufferSamples notdeproteinized (ifindicated ondatasheet)Cell/ tissue samplesnot sufficientlyhomogenizedToo many freezethawcyclesSamples containimpeding substancesSamples are too oldor incorrectly storedNot fully thawed kitcomponentsOut-of-date kit orincorrectly storedreagentsReagents sitting forextended periods oniceIncorrect incubationtime/ temperatureIncorrect amountsusedRefer to datasheet for detailsabout incompatible samplesUse the assay buffer provided(or refer to datasheet forinstructions)Use the 10kDa spin column(ab93349)Increase sonication time/number of strokes with theDounce homogenizerAliquot samples to reduce thenumber of freeze-thaw cyclesTroubleshoot and also considerdeproteinizing samplesUse freshly made samples andstore at recommendedtemperature until useWait for components to thawcompletely and gently mix prioruseAlways check expiry date andstore kit components asrecommended on the datasheetTry to prepare a fresh reactionmix prior to each useRefer to datasheet forrecommended incubation timeand/ or temperatureCheck pipette is calibratedcorrectly (always use smallestvolume pipette that can pipetteentire volume)12

For further technical questions please do not hesitate tocontact us by email (technical@abcam.com) or phone (select“contact us” on www.abcam.com for the phone number foryour region).13

UK, EU and ROWEmail: technical@abcam.comTel: +44 (0)1223 696000www.abcam.comUS, Canada and Latin AmericaEmail: us.technical@abcam.comTel: 888-77-ABCAM (22226)www.abcam.comChina and Asia PacificEmail: hk.technical@abcam.comTel: 108008523689 ( 中 國 聯 通 )www.abcam.cnJapanEmail: technical@abcam.co.jpTel: +81-(0)3-6231-0940www.abcam.co.jpCopyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.All information / detail is correct at time of going to print.15