Homogeneous Time Resolved Fluorescence (HTRF®) on Analyst ...

<strong>Homogeneous</strong> <strong>Time</strong> <strong>Resolved</strong> <strong>Fluorescence</strong> (HTRF®)on Analyst Multimode ReadersTable of ContentsIntroduction (1)Materials and Methods (2)Protocol (3)Introduction<strong>Time</strong>-<strong>Resolved</strong> <strong>Fluorescence</strong> Resonance Energy Transfer (TR-FRET) utilizes time-gateduorescence intensity measurements to quantitate molecular association or dissociation events.FRET is the non-radiative energy transfer between a uorophore and a chromophore. Thistransfer of energy is distance-dependent and requires an overlap of the donor emission andacceptor absorption spectra. <strong>Time</strong> resolved uorescence (TRF) measurements allow distinctionbetween short- and long-lived uorescent signals following excitation by a short flash of light.The immediate “prompt” uorescence dissipates in the rst few nanoseconds allowing a“delayed” measurement under conditions of nearly zero background. Consequently, thecombination of FRET-based assays and TRF measurements allow very sensitive detection ofbinding/dissociation events in a homogeneous format.TR-FRET can be applied to assays to detect the association of two molecules, as in receptorligandor protein-protein interactions, or when they are separated, as in the two ends of aprotease substrate after cleavage. The most commonly used labels are the long-livedlanthanide europium (donor) and the short-lived acceptor protein allophycocyanin (APC).Lanthanide metals are quenched in aqueous environments and thus require protection in theform of a proprietary cryptate.TR-FRET using HTRF® assay reagentsCommercially available reagents for TR-FRET can be obtained from CISbioInternational under the tradename HTRF ® . This application note discussesoptimized Analyst instrument settings and an evaluation of some plate types using amodel assay specially designed for validating instruments’ suitability for runningHTRF. The reagent kit uses biotin labeled europium cryptate (biot-K) and astreptavidin labeled allophycocyanin (SA-XL665). Results were obtained readingon either Analyst HT or Analyst GT Multimode Readers.Optimizing an HTRFDetection MethodParameters that were examined were:. • Optimization of adjustable instrument parameters for best sensitivityand signal:background ratio• Effects of different plate colors and well densities. • Effects of miniaturization on performance• Instrument to instrument variationMaterials and MethodsThe following materials are recommended for performing the HTRF assay.

Table 1. Equipment and materials:ItemAnalyst® Multimode Reader—one of thefollowing:• Acquest or Analyst AD or HT with at least v.2.0 CriterionHost software• ScreenStation with v. 2.5 ScreenPlaysoftware• Analyst GT with at least v. 3.0 AnalystHostsoftwareHTRF Reader Control Kit(includes reagents and 96 well ½ areamicroplate)SourcePlease contact your localMolecular Devices Corporationrepresentative for the instrumentbest suited to meet your needs.CIS bio P/N 62RCLPEACostar, 96 well black, ½ area Corning P/N 3694Costar, 96 well white, ½ area Corning P/N 3693Costar, 384 well white Corning P/N 3710Costar, 384 well black Corning P/N 3705Greiner, 1536 well white, high base Greiner P/N 782075Molecular Devices P/N 42-000-HE High Efficiency microplates, 96-well0117HE High Efficiency microplates, 384-well Molecular Devices P/N 0200-5201

Recommended Filter SetsFiltersTable 2. Recommended lter sets and dichroic:P/N forAnalyst AD,HT, orAcquestP/N forAnalyst GTHTRF filter set – all 4 pieces 42-000-0063 0200-6032330-80 nm excitation filter 42-000-0132 42-000-0132620 nm, donor emission filter 42-000-0064 42-000-0064665 nm, acceptor emission filter 42-000-0061 42-000-0061380 nm dichroic* mirror 42-000-0062 0200-6024* It is not recommended to use the 400 nm dichroic mirror included with the defaultEuropium filter set. When the 400 nm dichroic mirror is used with the HTRF filterset, the signal at 620 nm is lowered by ~ 30% and the signal to background ratio isadversely affected.Instrument Method SettingsTable 3 summarizes Molecular Devices’ recommended starting instrument parameters for HTRF assays:Table 3. HTRF assay settings:ParameterModeSwitch Methods byExcitation and emission ltersDichroic mirrorZ-heightAttenuatorDelay after ashSetting for Analyst AD, HT, orAcquestMulti-methodaPlatesee Table 2 abovesee Table 2 above1-2 mmOut50 µsecSetting for Analyst GTMulti-methodaWellsee Table 2 abovesee Table 2 above1-2 mmOut50 µsecIntegration time 400 µsec 400 µsecLamp Flash, maximum voltage Flash, maximum voltageFlashes per well 100<strong>Time</strong> between ashesRaw data unitsPlate settling timePMT setupTarget %CV per well1 X 10 msecCounts or Counts/sec10 msecdigitalN/A501 X 2 msecCounts or Counts/sec10 msecN/A0%a. Multi-method allows you to specify two previously dened TRF methods. This enables you togive a single read command that yields values for both donor and acceptor wavelengths. Inaddition, you can select automatic ratio calculations of these values or import the raw datainto SoftMax ® Pro 4.8 or higher for data analysis.

ProtocolReagent plates:Lyophilized vials of reagent were reconstituted and stored as instructed in the packageinsert. The reaction was incubated for 3 hours if the experimental reads could becompleted within 30 minutes but for 18 hours if it would be read over a period of >30minutes since the reaction has not reached equilibrium within 3 hours of incubation. Aplate incubated for 18 hours is stable for up to 24 hours. Each plate was laid out asillustrated in Table 4.Table 4. Plate lay-outABCDEFGH1 2 3 4 5 6 7 8 9 10 11 12620 nm Standard Standard Low High Buffer SA- Biot-KControl 0 (1/2 0 (1/2 control control blank XL665 blank(1/2 diluent, diluent, (1/2 (1/2 (1/2 blank (1/2control, ¼ biot- ¼ biot- low high diluent, (1/2 diluent,½ K, ¼ K, ¼ calib., calib, 1/2 diluent, ¼ biotrecon.)SA- SA- ¼ biot- ¼ biot- recon.) ¼ SA- K, ¼XL665) XL665) K, ¼ K, ¼XL665, recon)SA- SA-¼XL665) XL665) recon)The plates were covered and incubated at room temperature.Instruments:A donor and acceptor TRF method was set up then linked via a Multi-method in AnalystHT and Analyst GT. In Analyst HT the method was switched by plate (entire plate wasread in acceptor method, then entire plate was read in donor method). In Analyst GT, themethod was switched by well (acceptor then donor). The raw signal might varysignificantly from instrument to instrument due to differences in optical components(including lamps and filters) so data was analyzed using ratioed acceptor/donor signals.CalculationsThe fluorescence ratio associated with HTRF® readout is a correction method developedby CIS bio international and covered by patents for which CIS bio international. Rightsto use the patented equation are granted when a reagent kit is purchased from CIS bio.The calculations used for evaluating assay performance are shown in the following:Signal/Background (S/B) is calculated using the formula:S/B = signal 620 nm Control/signal 620 nm Buffer blank

Mean and % C.V. are calculated for each set of replicates then the following calculationsare done:RatioR= signal 665 nm/signal 620 nm * 1000Delta RatioDelta R =R CalX – R Standard 0(Cal X is either the low or high calibrator)Delta F%Delta F% = 100*(Delta R/R Standard 0)Optimizing settings in Standard 384 well black plateA standard black 384 well plate was set up as described in above Table 4. After 30minutes the plate was read in Analyst GT with varying z heights. The plate was also readat z height 1 mm with varying delay and integration times for the donor and acceptorreads. The analyzed results are shown below.Table 5. Optimizing Method SettingsZ heightAcceptortimings0 mm 50 usdelay, 400usintegration1 mm 50 usdelay, 400usintegration2 mm 50 usdelay, 400usintegration4 mm 50 usdelay, 400usintegration1 mm 50 usdelay, 150msintegration1 mm 50 usdelay, 400usDonortimings50 usdelay, 400usintegration50 usdelay, 400usintegration50 usdelay, 400usintegration50 usdelay, 400usintegration50 usdelay, 400usintegration200 usdelay, 800usS/B @620 nmLow CalRatioLowCalDelta FHighCalRatioHighCalDelta F54 364 20 1968 54852 289 2 2111 64544 366 0 2022 45154 330 -4 2185 53546 484 9 2646 49589 459 2 2867 537

integration integrationCIS bio Specs >40 >10 >500The best instrument settings for the standard 384 well plate appear to be zh = 0 mm, 50us delay and 400 us integration time for both the donor and acceptor readings.Comparison of HTRF results with different platesA standard 96 well plate was set up as described in Protocol Table 4. Each wellcontained 200 uL. After all reagents were combined, different volumes were transferredto different plate densities as shown in the Table 6 below.Table 6. Plates and Volumes testedPlate TypeFinalWellvolume½ Area 96 well black 100 uLStandard 384 well white 50 uLHE 96 well black20 uLHE 384 well black 10 uLStandard 1536 well white 6 uLThe plates were incubated, covered and sealed at room temperature for 18 hours. Usingthe optimum settings listed in Table 3 above, each plate was read in Analyst GT andAnalyst HT (except the 1536 well plate which was only read in GT).The analyzed results are shown below.Table 7. Comparison of microplates and volumesInstrument MicroplateAnalystGTAnalystHTAnalystGTAnalystHTAnalystGTAnalystHTAnalystGTAnalystHTAnalystGT½ Area 96 wellblack½ Area 96 wellblackStandard 384well whiteStandard 384well whiteHE 96 wellblackHE 96 wellblackHE 384 wellblackHE 384 wellblackStandard 1536well whiteS/B @620nmLow CalRatioLow CalDelta FHighCalRatioHighCalDelta F95 345 33 2320 797170 237 60 2730 1737127 288 26 2197 859137 316 33 2671 102280 336 22 2300 733134 230 39 2518 142264 402 29 2064 563119 145 -19 2128 108779 331 31 2174 759

CIS bio Specs >40 >15 >600Instrument-to-instrument variabilityA 384 well white plate was set up as described in Table 4 above and was incubated for 18hours at room temperature before being read on 3 Analyst GT using the settings in Table3. A summary of results is shown below:Table 8. Instrument to Instrument VariabilityInstrument 620 nmbackground(counts/sec)S/B @620nmLow CalRatioLow CalDelta FHighCalRatioHighCalDelta FAnalyst 1690 180 315 25% 2,212 782%GT #1Analyst 5137 219 386 29% 2,921 880%GT #2Analyst 6398 168 324 30% 2,333 836%GT #3CIS bio Specs >40 >15 >600Even though signal (counts/sec) varied between instruments by as much as 3.8 times, thesignal:background ratio and Delta F% were comparable and all surpassed specifiedlimits.