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1. Power off the instrument and install the LED cubes and imaging filter cubes see 3c Install the LED Cubes and Imaging Filter Cubes on page 17 in their defined locations Close the side access door Leave Gen5 open with the Reader Setup dialog onscreen while performing the next steps Power on the instrument After the self test the instrument will beep and the eject button LED will be red indicating that the objectives must be calibrated Press the carrier eject button to stop the beeping Click Auto Calibration Note that this process can take up to 15 minutes on an instrument with six objective installed Phase contrast components are calibrated in the factory before shipment You do not need to run the Phase Ring Configuration and Calibration routines at this time BioTek Instruments Inc 14 Install the Imager Module 17 3a Setting the Objective LED Cube and Imaging Filter Cube Configuration When you install an LED cube an imaging filter cube or an objective you must set the cubes and objectives configuration before physically changing or installing them If you physically install an LED cube an imaging filter cube or an objective before setting the new configuration the instrument may fail ts self test 1 From the main Gen5d screen click System gt Instrument Configuration Select the Cytation 5 click View Modify gt Setup and select the Imaging Configuration tab 3 In the Objective Con
2. Click Plate gt Read Plate save the experiment then click OK When prompted by Geno rotate the plate 180 degrees in the carrier When the read is finished analyze the results as described below Analyze the Results 1 4 5 Calculate the Mean focus height for each set of ten reads in the Normal position in wells A1 A12 H1 and H12 Calculate the Mean focus height for each set of ten reads in the Turnaround position in wells A1 A12 H1 and H12 Compare the Mean values for each well in its Normal and Turnaround positions by performing these calculations 1 A1 Normal Mean H12 Turnaround Mean H12 Normal Mean A1 Turnaround Mean 1 A12 Normal Mean H1 Turnaround Mean H1 Normal Mean A12 Turnaround Mean For A1 H12 e Calculate the Mean Delta um step a results step b result 2 e Calculate the Carrier Tilt Mean Delta 25400 um e The Carrier Tilt must be less than 0 004 to Pass For A12 H1 e Calculate the Mean Delta um step c results step d result 2 e Calculate the Carrier Tilt Mean Delta 25400 um e The Carrier Tilt must be less than 0 004 to Pass AutoFocus Test The AutoFocus test confirms the imaging system s ability to repeatedly focus on a known target 1 Place the imaging qualification plate on the carrier with well A1 facing up in the right rear corner of the carrier Cytation 5 60 Instrument Testing 2 If you have not already done so create the proto
3. Bottom Mono Fluorescence Top Filter Fluorescence Top Mono Fluorescence Dispense Read for models with the dual reagent dispense module Both injectors dispense to standard height 6 12 24 48 96 Plate Type and 384 well microplates Detection Method Absorbance Fluorescence FI FP TRF Luminescence Imaging well mode only Volume Range 5 1000 uL with a 5 20 uL tip prime dead eee lt 1100 uL with dead volume recovery function back flush Injection Speeds 225 250 275 300 uL second 1 uL or 2 0 whichever is greater lt 2 0 for volumes of 50 200 uL lt 4 0 for volumes of 25 49 uL Precision lt 7 0 for volumes of 10 24 uL lt 10 0 for volumes of 5 9 UL BioTek Instruments Inc Absorbance Specifications 105 Absorbance Specifications Wavelength Range 230 to 999 nm Wavelength Bandpass lt 4 nm 230 285 nm lt 8 nm gt 285 nm Measurement Range 0 000 to 4 000 OD Minimum kinetic interval 450 nm lt 20 seconds sweep mode 96 well microplate Plate In Plate Out Speed lt 35 seconds 450 nm sweep mode 96 well microplate Accuracy Linearity Repeatability Specifications apply from 250 999 nm 200 uL 96 well microplates Accuracy tested with certified neutral density glass 96 well plate normal read speed 0 2 OD 1 0 010 OD delay after plate movement 100 ms 2 2 5 OD 3 0 010 OD delay after plate movement 100 ms 384 well plate normal read speed 0 2
4. hazards Warning Electrical Grounding Never use a plug adapter to connect primary power to the external power supply Use of an adapter disconnects the utility ground creating a severe shock hazard Always connect the power cord directly to an appropriate receptacle with a functional ground Warning Service Only qualified technical personnel should perform service procedures on internal components Warning Accessories Only accessories that meet the manufacturer s specifications shall be used with the instrument Warning Lubricants Do not apply lubricants to the microplate carrier or carrier track Lubricant on the carrier mechanism or components in the carrier compartment will attract dust and other particles which may obstruct the carrier path and cause the instrument to produce an error Warning The instrument with all available modules weighs up to 80 Ibs 36 3 kg Use two people when lifting and carrying the instrument Warning Liquids Avoid spilling liquids on the instrument fluid seepage into internal components creates a potential for shock hazard If a spill occurs while a program is running abort the program and turn off the instrument Wipe up all spills immediately Do not operate the instrument if internal components have been exposed to fluid Contact BioTek TAC for assistance Cytation 5 vi Preface Warning Unspecified Use Failure to operate the equipment according to the guidelines and saf
5. s Technical Assistance Center BioTek Instruments Inc Fluorescence Liquid Tests 77 e Are the solutions fresh Discard the plate and any open unused test solutions after seven days e Are the excitation emission filters clean Are they in the proper locations and in the proper orientation in the filter cube e Ifthe Corners Test continues to fail the hardware may be misaligned Contact BioTek TAC e Are you using new clean plates If the base of a clear bottom plate is touched clean the entire base with alcohol 95 ethanol and then wipe with a lint free cloth Before placing the plate in the instrument blow the bottom of the plate with an aerosol duster If the test fails again the optical probe s may need to be cleaned Contact BioTek s Technical Assistance Center for instructions e Review the pipetting instructions to verify the plate was correctly prepared e Does the Plate Type setting in the Gen5 protocol match the plate you used e For injector models spilled fluid inside the reader may be fluorescing which can corrupt your test results If you suspect this is a problem contact BioTek TAC e When testing Fluorescence Polarization capability using a solid black plastic microplate if the standard deviation for the buffer wells is too high try moving the buffer wells to another column With some black plastic plates the wells in the center of the plate may be slightly distorted due to the plate molding process
6. 4 on the filter slide 3 Use the 3 32 hex wrench to screw the LED cube onto the filter slide 4 Place the imaging filter cube on top of the LED cube you installed and use the hex wrench to screw it to the LED cube 5 Insert the LED cube s wire clip into the socket on the carrier BioTek Instruments Inc 14 Install the Imager Module 19 connect the LED wires to their sockets Cytation 5 20 Installation gt wire clip r 6 Slide the filter slide back into the instrument 7 Close the side access door Run Auto Calibration After you have physically installed the LED cubes imaging filter cubes and objectives you must run Auto Calibration before you can use the imaging module This process can take up to 15 minutes on an instrument with six objectives installed 1 Turn on your instrument and allow the self test to run The instrument will beep indicating that the self test has failed 2 Press the carrier eject button to stop the beeping 3 If the Reader Setup dialog is not already visible on your screen go to System gt Instrument Configuration select Cytation 5 then click View Modify gt Setup 4 On the Instrument Configuration tab click Auto Calibration 5 If you have a 40X or 60X objective installed Gen5 prompts you to place the objective setup plate PN 1222531 on the carrier After you place the setup plate on the carrier click OK After the calibration procedure is fin
7. 5 dye powder into a weigh boat Rinse the contents into a 1 liter volumetric flask Add 0 5 mL of Tween 20 or 5 mL of BioTek s wetting agent Fill to 1 liter with DI water cap and shake well The solution should measure approximately 2 000 OD when using 200 uL in a flat bottom microwell Procedure 1 Using freshly prepared stock solution Solution A or B prepare a 1 2 dilution using deionized water one part stock one part deionized water the resulting solution is a 1 2 dilution Pipette 200 uL of the concentrated solution A or B into the first column of wells in the microplate Pipette 200 uL of the diluted solution into the second column of wells Using Gen5 read the plate five times at 405 nm using the Normal read mode single wavelength no blanking Save the data after each read Normal plate position Without delay rotate the plate 180 degrees so that well A1 is in the H12 position Read the plate five more times saving the data after each read Turnaround plate position Print ten sets of raw data or export them to an Excel spreadsheet BioTek Instruments Inc Fluorescence Liquid Tests 67 Analyze the Results e The plate is read five times in Normal position at 405 nm Calculate the Mean OD and Standard Deviation of those five reads for each well in columns 1 and 2 e For each well in columns 1 and 2 calculate the Allowed Deviation using the repeatability specification for a 96
8. 74 Instrument Testing Time Resolved Fluorescence TRF Test e Pipette 200 uL of deionized water into wells A6 C6 e If you have not already done so shake the vial of 20 pM europium suspension vigorously for 30 seconds prior to pipetting Alternatively sonicate or vortex the vial e Pipette 200 uL of the 20 pM europium suspension Eu into well A8 T Q TIM OD O D gt Results Analysis Corners Test 1 Calculate the Mean of the 12 wells containing the 3 3 nM SF test solution A1 A3 A10 A12 H1 H3 and H10 H12 2 Calculate the Standard Deviation for the same 12 wells 3 Calculate the CV Standard Deviation Mean 100 The CV must be lt 3 0 to pass Sensitivity Test 1 Calculate the Mean and Standard Deviation of the 16 reads for each of the buffer wells C9 D9 E9 2 Among the three buffer wells find the Median Standard Deviation and corresponding Mean 3 Calculate the Mean for the 16 reads of the SF Concentration well D7 4 Calculate the Signal to Noise Ratio SNR using the Mean SF Concentration Buffer Median STD with its corresponding Buffer Mean SF Mean Buffer Mean 3 Buffer STD 5 Calculate the Detection Limit in pM using the known concentration value of SF and the Calculated SNR 1000 SNR BioTek Instruments Inc Fluorescence Liquid Tests 75 Filter Based Fluorescence System Optic Probe To pass the Detection Limit must be less than or equal to Top 10 pM
9. 86 F Note Performance measurements including detection limits were verified up to 25 C 77 F Humidity 10 to 85 relative humidity non condensing 24 volt external power supply compatible with 100 240 V 10 50 60 Hz Power Consumption 130W maximum Temperature control ranges from 4 C over ambient to 65 C Temperature variation 0 5 C across the plate 37 C tested with Innovation Instruments Inc temperature test plate Environment Power Supply Incubation Top and bottom incubation controlled via software adjustable gradient For models with the alpha laser module Alpha laser module operation is disabled above an internal instrument temperature of 35 C BioTek Instruments Inc Plate Shaking General Specifications 103 Linear Amplitude 1 mm to 6 mm in 1 mm steps Frequency 18 Hz to 6 Hz Orbital Slow Amplitude 1 mm to 6 mm in 1 mm steps Frequency 10 Hz to 3 Hz Orbital Fast Amplitude 1 mm to 6 mm in 1 mm steps Frequency 14 Hz to 5 Hz Double Orbital Slow Amplitude 1 mm to 6 mm in 1 mm steps Frequency 10 Hz to 3 Hz Double Orbital Fast Amplitude 1 mm to 6 mm in 1 mm steps Frequency 14 Hz to 5 Hz Frequency is based on the amplitude selected Cytation 5 104 Specifications Dispense Read Specifications Maximum Delay between End of Dispense and Beginning of Read 96 384 well plates default probe heights
10. Corriente continua y corriente alterna Corrente continua e corrente alternata Earth ground terminal Borne de terre Erde Betriebserde Borne de tierra Terra di funzionamento Protective conductor terminal Borne de terre de protection Schultzleiteranschluss Borne de tierra de protecci n Terra di protezione gt Warning risk of crushing or pinching Attention risque d crasement et pincement Warnen Gefahr des Zerquetschens und Klemmen Precauci n riesgo del machacamiento y sejeci n Attenzione rischio di schiacciare ed intrappolarsi Warning hot surface Attention surface chaude Vorsicht heiBe Oberfl che Precauci n superficie caliente Attenzione superfice calda Laser radiation Do not stare into beam Rayonnement laser Ne pas regarder dans le faisceau Laserstrahlung nicht in den strahl blicken Radiaci n de laser No mire fijamente al rayo Radiazione di laser Non stare nel fascio Warning potential biohazards Attention risques biologiques potentiels Warnung Moegliche biologische Giftsoffe Atenci n riesgos biol gicos Attenziones rischio biologico Caution refer to accompanying documents Attention voir documents d accompanement Achtung siehe Begleitpapiere Atencion vease los documentos incluidos Attenzione consultare la doc annessa xii Preface On Supply Consult instructions for use Marche alimentation Consulter la notice d emploi Ein Verbind
11. Cytation 5 12 Installation Desktop Computer Install the FireWire Card To avoid electrostatic discharge and damage to internal components ground yourself by using wrist grounding straps or by touching a metal surface on the computer s chassis The following directions provide general steps for installing a PCI Express card Talk to your company s IT representative for assistance with these steps For more detailed help contact BioTek TAC 1 Turn off your computer then remove the outside case Depending on the computer remove either one side of the tower or the entire cover 2 Locate the PCI Express slot Open the card retainer and remove any existing graphics card if necessary or blank port located in the PCI Express slot pue mu AE a 3 Insert the PCI Express card aligning it with its slot 4 Press the card firmly into place and secure the card with a locking rail or screws 5 Replace your computer s outside case then power on the computer BioTek Instruments Inc 14 Install the Imager Module 13 Laptop Computer Install the FireWire Card 1 Remove the PCI Express port cover or any existing PCI Express card from your laptop FireWire card slot 2 Touch a metal object to discharge any static electricity 3 Remove the PCI Express card from the packaging 4 Attach the spacer if required to the FireWire PCI Express card The spacer does not fit tightly Ensure that it does not f
12. G Factor Perpendicular HPR Signal 1000 Parallel HPR Signal Mean G Factor Perpendicular HPR Signal 6 Calculate the Mean PHPR in mP To pass the Mean PHPR ABUSES must be greater than Top with 510 nm dichroic mirror 340 mP 7 Calculate the Polarization value in mP for each LPR well PLPR Parallel LPR Signal Mean G Factor Perpendicular LPR Signal 1000 Parallel LPR Signal Mean G Factor Perpendicular LPR Signal Calculate the Standard Deviation of the PLPR in mP Optic Probe To pass the Standard Deviation 7 of the PLPR must be less than Time Resolved Fluorescence TRF Test 1 Calculate the Mean and Standard Deviation of the 16 reads for each of the buffer wells A6 B6 C6 2 Among the three buffer wells find the Median Standard Deviation and corresponding Mean 3 Calculate the Mean for the 16 reads of the Eu Concentration well A8 4 Calculate the Signal to Noise Ratio SNR using the Mean Eu Concentration and Buffer Median STD with its corresponding Buffer Mean Eu Mean Buffer Mean 3 Buffer STD 5 Calculate the Detection Limit in fM 20000 Mean Eu Mean DI water 3 Standard Deviation DI water Optic Probe To pass the Detection Limit must be less than or equal to Top with 400 nm dichroic mirror 250 fM Troubleshooting If any tests fail please try the following suggestions If the test s continue to fail print the results and contact BioTek
13. If you are using PBS prepare the solution e Optional but recommended Using a 0 45 micron filter filter 200 mL of deionized or distilled water e Follow the manufacturer s instructions on the PBS packaging to create 200 mL dissolving the necessary amount of PBS into the filtered water e Stir the solution preferably using a stir table until the PBS is completely dissolved e Check the pH it should be between 7 2 and 7 6 at 25 C 2 Prepare the sodium fluorescein stock solution e Add 2 0 mL of the buffer solution to the 1 mg Sodium Fluorescein SF vial This yields a 1 3288 mM stock solution e Ensure that the dye has completely dissolved and is well mixed 3 Carefully prepare the dilutions Label each with SF and the concentration Mix This SF Solution 0 53 mL of 1 3288 mM stock 13 47 mL 50 2 uM solution 110 uL of 50 2 uM SF 13 89 mL 400 nM 3 5 mL of 400 nM SF 10 5 mL 100 nM 0 46 mL of 100 nM SF 13 54 mL Corners Test Sensitivity Linearity Tests 4 24 mL of 3 3 nM SF 9 76 mL 1 nM BioTek Instruments Inc Fluorescence Liquid Tests 71 Fluorescence Polarization FP Test The recommended test solutions are available from Invitrogen Corporation or from BioTek They do not require additional preparation Time Resolved Fluorescence TRF Test The recommended test solutions are available from Invitrogen Corporation or from BioTek e Shake the FluoSpheres container vigorously for 30 seconds pr
14. OD 2 0 010 OD delay after plate movement 100 ms 2 2 5 OD 5 0 010 OD delay after plate movement 100 ms 96 well and 384 well plate sweep read speed 0 1 OD 1 0 010 OD Cytation 5 106 Specifications Accuracy Linearity Repeatability Linearity by liquid dilution 96 well plate normal read speed 0 2 OD 1 0 010 OD delay after plate movement 100 ms 2 2 5 OD 3 0 010 OD delay after plate movement 100 ms 384 well plate normal read speed 0 2 OD 2 0 010 OD delay after plate movement 100 ms 2 2 5 OD 5 0 010 OD delay after plate movement 100 ms 96 well and 384 well plate sweep read speed 0 1 OD 1 0 010 OD Repeatability tested with certified neutral density glass measured by one standard deviation 8 measurements per data point 96 well and 384 well plate normal read speed 0 2 OD 1 0 005 OD delay after plate movement 100 ms 2 2 5 OD 3 0 005 OD delay after plate movement 100 ms 96 well and 384 well plate sweep read speed 0 1 OD 2 0 010 OD Take3 Plate Detection Limit 260 nm dsDNA lt 5 ng pL BioTek Instruments Inc Fluorescence Specifications Mono Based 107 Fluorescence Specifications Mono Based The Cytation 5 measures fluorescence with monochromators from the top and bottom of 6 to 384 well plates All detection limit DL requirements are measured by the two point method whi
15. and this can affect the standard deviation e The Read steps in the protocols use the Gen5 Automatic Gain Adjustment feature to determine optimum sensitivity values for the plate If an Auto Gain Result value is outside the range of 50 200 this may indicate a problem If the value is less than 50 The stock solution dilution concentrations may be too high Try creating fresh solutions dilutions and rerun the test using a new clean plate If all of the tests are passing but the Gain value is low a PMT in your reader may just be very sensitive Contact BioTek s Technical Assistance Center to confirm that this may be the case If the value is greater than 200 The stock solution dilution concentrations may be too low Try creating fresh solutions dilutions and rerun the test using a new clean plate For injector models spilled fluid inside the reader may be fluorescing which can corrupt your test results If you suspect this is a problem contact BioTek TAC The PMTs or optical path s may be deteriorating or the optics or other hardware may be misaligned Contact BioTek s Technical Assistance Center Gen5 Protocol Reading Parameters The information in the following tables represents the recommended reading parameters It is possible that your tests will require modifications to some of these parameters such as the Plate Type see Troubleshooting Tips on page 76 Cytation 5 78 Instrument Testing The Plate
16. and washer located between the emission filter positions 3 Carefully lift the filter cube top from the cube 4 Wearing linen or cloth gloves grasp the mirror by its edges and lift it out of the cube The mirrors are seated on a shelf in the bottom of the cube and are not secured in place Cytation 5 48 Maintenance 5 Wet absorbent towels such as Kimwipes not lens paper with anhydrous reagent grade ethanol Wear gloves or use enough toweling so that solvents do not dissolve oils from your hands that can seep through the toweling onto the coated surface 6 Drag the trailing edge of the ethanol soaked Kimwipe across the surface of the mirror moving in a single direction A minimal amount of pressure can be applied while wiping However too much pressure will damage the mirror 7 Use the magnifying glass to inspect the surface if debris is still visible repeat with a new Kimwipe 8 To replace the mirror hold it by its edges turn it so that its label is face up and readable and place it on the shelf in the filter cube Filter cube label Mirror labels 9 Place the filter cube top back onto the cube and replace the screw and washer 10 When finished reinstall the filter cube in the reader Inspect Clean Excitation and Emission Filters Applies only to Cytation 5 models with filter based fluorescence capabilities Laboratory air is used to cool the flash bulb and the filter cubes can become dusty
17. change the filters and mirrors yourself Note These cubes are not to be confused with the imaging LED cubes and filter cubes Applies to models with the dispense module The syringes may require replacement over time The tubing and injectors require cleaning at regular intervals Applies to models with the imaging module The imaging system comprising a CCD camera objectives LED cubes and filter cubes allows you to run experiments with imaging reads as well as view images in live mode Cytation 5 28 Getting Started Filter Cube The Cytation 5 is equipped with a filter cube that contains excitation and emission filters mirrors and if required polarizing filters Each filter cube contains two filter sets each of which contains one excitation filter one mirror and one emission filter The filter cube is accessed through a hinged door on the front of the instrument Synergy H1 filter cubes are interchangeable with the filter cubes for the Cytation 5 Do not open the door to access the filter cube during instrument operation Doing so may result in invalid data Define the Filters Gen keeps track of each cube s contents and communicates this information to the instrument during operation You must define characteristics for your filter cube s in the Gen Filter Cube Table System gt Instrument Configuration gt Setup e Select Band Pass Long Pass or Short Pass as appropriate for each filter type e Band P
18. flow in and out of the instrument The reader is sensitive to extreme environmental conditions Avoid the following e Excessive humidity Condensation directly on the sensitive electronic circuits can cause the instrument to fail internal self checks The humidity must be in the range of 10 85 non condensing e Excessive ambient light Bright light may affect the reader s optics and readings reducing its linear range e Dust Readings may be affected by extraneous particles such as dust in the microplate wells A clean work area is necessary to ensure accurate readings e Vibration The instrument should be installed in a vibration free environment Be sure to position the instrument away from other devices that could potentially create vibration during the read process If you are installing a BioStack for operation with the Cytation 5 you may wish to seat the instruments in their alignment plates now Refer to the stacker s operator s manual for more information BioTek Instruments Inc 4 Select an Appropriate Location 5 Installing Instruments with the Isolation Table Cytation 5 models with the imaging module can be used with an isolation table which helps to eliminate vibration during image reads Do not use the isolation table when operating the Cytation 5 with a microplate stacker Store the isolation table in a clean dry location 1 Remove the four corner clips from the isolation table Isolation
19. imaging capability Used canned air to blow debris from the aperture on the carrier Do not wipe with liquid which can seep inside aperture s glass plates and impact imaging reads Cytation 5 46 Maintenance AA E Y Wipe all exposed surfaces of the dispense module if used Wipe all exposed surfaces of the gas controller module if used If detergent was used wipe all surfaces with a cloth moistened with water Use a clean dry cloth to dry all wet surfaces ae Models with a dispenser If the Tip Priming Trough overflows or other spills occur inside the instrument wipe the carrier and the surface beneath the carrier with a dry cotton cloth The internal chamber and probes are not customer accessible If overflow is significant contact BioTek s Technical Assistance Center with any questions about your particular model Inspect Clean Mirrors Applies only to Cytation 5 models with filter based fluorescence capabilities We recommend inspecting cleaning the mirrors and polarizing filters if equipped annually especially if the filter cube has been opened or changed These optical elements are delicate and should be handled as carefully as possible The glass and anti reflective AR coated surfaces will be damaged by any contact especially by abrasive particles In most cases it is best to leave minor debris on the surface However if performance indicators or obvious defects in the mirror
20. pieces of opaque PTFE Teflon tubing connected to stainless steel probes on one end and threaded fittings on the other end 3 Solenoid valves allow the fluid drawn from the supply bottles by the syringe pumps to flow into the outlet tubes 4 Outlet tubes transport fluid from the syringes into the instrument through the tubing ports on the Cytation 5 s top cover The outlet tubes are opaque PTFE tubes with threaded fittings on each end Dispense Module Components and Materials Composition Continuous contact with harsh chemicals is not recommended Always rinse the fluid path with deionized water after contact with any strong acid base or solvent Cytation 5 30 Getting Started Components Material Composition Tubing syringe fittings PTFE polytetrafluoroethylene Injector tips 315 stainless steel Injector body PVC polyvinyl chloride Priming plate and trough Polyproylene Valve diaphragms Ethylene propylene EPDM Valve body PEEK polyether ether ketone Syringe barrel Borosilicate glass Priming the Injector System Before running a dispense assay prime the system with the reagent or dispensing fluid In addition tip priming can be performed at the start of an assay and sometimes just before each dispense to a well The tip prime compensates for any fluid loss at the injector tip due to evaporation since the last dispense All priming activities are controlled via Gen5 If the injector system is not primed adequately
21. test dark gray wells e Pipette 200 pL of the 1 nM SF solution into well D7 e Pipette 200 pL of the buffer solution into wells C9 D9 and E9 For the Linearity test wells C1 F5 e Use a multichannel pipette with just four tips installed e Pipette 150 pL of buffer solution into wells C2 F5 Discard the tips e Pipette 150 pL of the 1 nM SF solution into wells C1 Fl Pipette 150 pL of the 1 nM SF solution into wells C2 F2 Mix the wells using the pipette Do not discard the tips BioTek Instruments Inc Fluorescence Liquid Tests 73 e Aspirate 150 pL from wells C2 F2 and dispense into wells C3 F3 Mix the wells using the pipette Do not discard the tips e Aspirate 150 pL from wells C3 F3 and dispense into wells C4 F4 Mix the wells using the pipette Do not discard the tips e Aspirate 150 pL from wells C4 F4 and dispense into wells C5 F5 Mix the wells using the pipette Do not discard the tips e Aspirate 150 pL from wells C5 F5 and discard the tips OO COCO AO mE J la ete ate Cl A OE A CBUF CBUF CBUF CBUF CBUF CBUF CBUF CBUF CBLIF applies only to the Hellma Quartz plate E Tl G Fluorescence Polarization FP Test e Pipette 200 uL of the green polarization buffer BUF into wells A6 H6 e Pipette 200 uL of the green high polarization reference HPR into wells A7 B7 e Pipette 200 uL of the green low polarization reference LPR into wells A8 H8 Cytation 5
22. test continues to fail the laser may not be firing Contact BioTek TAC Gen5 Protocol Reading Parameters The information in the following table represents the recommended reading parameters It is possible that your tests will require modifications to some of these parameters such as Plate Type or Gain value see Troubleshooting Tips above The Plate Type setting in the Gen5 protocol should match the plate you are actually using Cytation 5 AlphaTest_Crosstalk prt Plate Type Costar 96 well white opaque Detection Method Alpha Read Type Endpoint BioTek Instruments Inc Dispense Module Tests 95 Dispense Module Tests This section applies only to models with the dispenser Required Materials e Absorbance reader with capability of reading at 405 630 and 750 nm The reader must have an accuracy specification of 1 0 0 010 OD or better and a repeatability specification of 1 0 0 005 OD or better The Cytation 5 may be used if it is equipped with absorbance capabilities and has passed the Absorbance Plate Test or Absorbance Liquid Test 1 e Microplate shaker if the absorbance reader does not support shaking e Precision balance with capacity of 100 g minimum and readability of 0 001 g e 50 200 uL hand pipette and disposable tips e Deionized water e Supply bottles e 250 mL beaker e New 96 well clear flat bottom microplates e BioTek s Green Test Dye Solution PN 7773003 undiluted or one of the
23. threshold 100 Vibration Detection Options CV Threshold ite Images to average 5 Horizontal offset from center of well Vertical offset from center of well Single image per well Enabled Absorbance Liquid Test This test confirms the reader s ability to perform to specification with liquid samples If the test passes then the lens placement and optical system cleanliness are proven Materials e New 96 well clear flat bottom microplate Corning Costar 3590 recommended e Stock Solution A or B which may be formulated by diluting a dye solution available from BioTek A or from the ingredients listed below B Solution A e BioTek QC Check Solution No 1 PN 7120779 25 mL PN 7120782 125 mL e Deionized water Cytation 5 66 Instrument Testing e 5 mL Class A volumetric pipette e 100 mL volumetric flask 1 Pipette a 5 mL aliquot of BioTek QC Check Solution No 1 into a 100 mL volumetric flask 2 Add 95 mL of DI water cap and shake well The solution should measure approximately 2 000 OD when using 200 uL in a flat bottom microwell Solution B e Deionized water e FD amp C Yellow No 5 dye powder typically 90 pure e Tween 20 polyoxyethylene 20 sorbitan monolaurate or BioTek wetting agent PN 7773002 a 10 Tween solution e Precision balance with capacity of 100 g minimum and readability of 0 001 g e Weigh boat e 1 liter volumetric flask pe Wee Weigh out 0 092 g of FD amp C Yellow No
24. well plate 1 0 005 OD from 0 000 to 2 000 OD Mean 0 010 0 005 For each well its standard deviation should be less than its allowed deviation e The plate is read five times in the Turnaround position at 405 nm Calculate the Mean OD of those reads for each well in columns 11 and 12 e Perform a mathematical comparison of the Mean values for each microwell in its Normal and Turnaround positions that is compare Al to H12 A2 to H11 B1 to G12 H2 to A11 To pass the test the differences in the compared mean values must be within the accuracy specification for a 96 well microplate 1 0 0 010 OD from 0 000 to 2 000 OD Repeatability Specification 1 0 005 OD from 0 000 to 2 000 OD 3 0 0 010 OD from 2 000 OD to 2 500 OD Fluorescence Liquid Tests Required Materials All Tests e Deionized or distilled water e Various beakers graduated cylinders and pipettes e 95 ethanol for cleaning clear bottom plates e Aluminum foil e Optional but recommended 0 45 micron filter e Optional Black polyethylene bag s to temporarily store plate s e Gen5 protocols listed below and described on page 77 For the Filter Based Fluorescence System Corners Sensitivity and Linearity tests using the top Cytation 5_FI_T_SF prt optics Cytation 5_FP prt Fluorescence Polarization test Cytation 5_TRF prt Time Resolved Fluorescence test Cytation 5 68 Instrument Testing For the Monochromator Based Fl
25. 00 mm O UN cr y MD 5 D y O D Read Height Read Step 4 Detection Method Fluorescence Read Type Endpoint Linearity Read C1 F5 1 Green EX 485 20 nm EM 528 20 nm Top 510 nm Auto Scale to High Wells C1 30000 Read Wells Filter Set Filters Optics Position O UN cr y D 5 D y O D Cytation 5 80 Instrument Testing Normal Delay after plate movement 350 msec Read Speed Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Cytation 5_FP prt This procedure contains one Read step using filters with Fluorescence Polarization enabled inside a Plate Mode block Filter Sets 1 filter cube Filters EX 485 20 nm EM 528 20 nm Optics Position Top 510 nm Gain Auto Scale to high Wells A8 10000 Normal Delay after plate movement 350 msec Measurements per data point 60 Lamp Energy Low Read Speed Read Height Cytation 5_TRF prt Parameter Default Setting Plate Type Costar 96 well white opaque Read Step 1 Detection Method Time resolved fluorescence BioTek Instruments Inc Fluorescence Liquid Tests 81 Read Type Endpoint Run Time 00 00 15 Kinetic Loop Interval 00 00 01 Reads 16 Step Label Sensitivity Read 1 filter cube Filters EX 360 40 nm 620 40 nm Optics Position Top 400 nm Auto Scale to High Wells A8 30000 Normal Delay after plate movement 100 msec Measurements per data point 50 Lamp E
26. 8032028 adapter Step Label O UN cor y MD 5 D y O D Read Step 1 Read Type Endpoint Cytation 5 90 Instrument Testing Step Label Reference well A2 Integration Time 0 10 00 MM SS ss 350 msec Standard 1 00 mm Delay after plate movement Dynamic Range Read Height Read Step 2 Detection Method Read Type Luminescence Endpoint Background Read Wells F1 G12 E UI O G 0 0 g O 5 pS Eo y O a D 5 0 10 00 MM SS ss 350 msec Standard 1 00 mm Integration Time Delay after plate movement Dynamic Range Read Height Read Step 3 Detection Method Read Type Luminescence Endpoint Battery Check A7 A8 p Label Read Wells 0 01 00 MM SS ss 350 msec Standard 1 00 mm Integration Time Delay after plate movement Dynamic Range Read Height G 0 Y O sS lt ae o D 5 ta cr gt BioTek Instruments Inc Luminescence Test 91 Cytation 5 F LumTest_Glowell prt Read Wells A8 H11 Filter Sets 1 Open Cytation 5 M LumTest_Glowell prt Read Wells A8 H11 Cytation 5 92 Instrument Testing Alpha Detection Test This section applies only to models with the alpha module For BioTek Service Personnel only An alternate method for conducting this test is to follow the Alpha Laser Calibration instructions in the Cytation 5 Service Manual PN 1321023 This calibration requires the use of the EM Mono Efficien
27. Cytation 5 Cell Imaging Multi Mode Reader Instructions for Use BioTek Instruments Inc January 2015 2015 PN 1321022 Revision A ii Preface Notices BioTek Instruments Inc Highland Park P O Box 998 Winooski Vermont 05404 0998 USA All Rights Reserved 2015 BioTek Instruments Incorporated No part of this publication may be reproduced transcribed or transmitted in any form or by any means electronic or mechanical including photocopying and recording for any purpose other than the purchaser s use without written permission of BioTek Instruments Inc Trademarks BioTek is a registered trademark and Cytation Gen5 BioStack and Take3 are trademarks of BioTek Instruments Inc Glowell is a trademark of LUX Biotechnology Ltd Harta is a trademark of Harta Instruments Microsoft Windows and Excel are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries All other trademarks are the property of their respective holders Restrictions and Liabilities Information in this document is subject to change and does not represent a commitment by BioTek Instruments Inc Changes made to the information in this document will be incorporated in new editions of the publication No responsibility is assumed by BioTek for the use or reliability of software or equipment that is not supplied by BioTek or its affiliated dealer
28. Directive 2002 96 EC on waste electrical and electronic equipment WEEE or local ordinances Directive 98 79 EC In Vitro Diagnostics if labeled for this use e Product registration with competent authorities e EN 61010 2 101 Particular requirements for in vitro diagnostic IVD medical equipment e Traceability to the U S National Institute of Standards and Technology NIST Electromagnetic Interference and Susceptibility USA FCC CLASS A RADIO AND TELEVISION INTERFERENCE NOTE This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at their own expense In order to maintain compliance with FCC regulations shielded cables must be used with this equipment Operation with non approved equipment or unshielded cables is likely to result in interference to radio and television reception Canadian Department of Communications Class A This dig
29. Emission 528 20 510 nm mirror DL lt 0 16 ng mL 0 91 nM typical solution of Methylumbelliferone in CBB Excitation 360 40 Emission 460 40 400 nm mirror Time Resolved Fluorescence DL Europium lt 250 fM 100 fM typical Excitation 360 40 nm Emission 620 40 nm 400 nm mirror Integration time 20 to 2000 us Delay O to 2000 us Granularity 1 us step Fluorescence Polarization 5 mP standard deviation at 1 nM Sodium Fluorescein Excitation 485 20 nm Emission 528 20 nm 510 nm mirror Excitation range 400 to 700 nm Emission range 400 to 700 nm BioTek Instruments Inc Luminescence Specifications 109 Luminescence Specifications The Cytation 5 measures luminescence from the top of 6 to 384 well plates The following requirements apply to 96 well plates with 200 uL well at room temperature Production testing is performed using a Harta plate Luminescence DL 75 amol well 30 amol typical with low noise PMT 500 amol well with red shifted PMT Imaging Specifications The Cytation 5 imaging specifications are based on using an NIH 3T3 plate 10 000 wells per cell GFP stain Costar 3603 black sided plastic bottom plate Read Speed At 20X Autofocus on 96 well plate 1 image per well lt 10 minutes Cytation 5 110 Specifications BioTek Instruments Inc
30. Fluorescence Polarization capability e Pipette the solutions for the FP test into the same plate as used in step 3 e Create an experiment based on the Cytation 5_FP prt protocol Read the plate and then save the experiment 5 Perform the Corners Sensitivity Linearity tests for the monochromator based fluorescence system e Create experiments based on the Cytation 5_M_FI_T_SF prt for the top optics and Cytation 5_M_FI_B_SF prt for the bottom optics e Read the plate and then save the experiment 6 To test the Time Resolved Fluorescence capability e Pipette the solutions for the TRF test into a new 96 well solid white plate e Create an experiment based on the Cytation 5_TRF prt protocol Read the plate and then save the experiment Pipette Maps Seal the plates with foil or store them in black polyethylene bags until use When using a clear bottom plate if the base of the plate is touched clean the entire base with alcohol 95 ethanol and then wipe with a lint free cloth Before placing the plate in the instrument blow the bottom of the plate with an aerosol duster Perform these steps carefully and refer to the grid on the next page For the Corners test light gray wells e Pipette 200 pL of the 3 3 nM SF solution into wells A1 A3 A10 A12 H1 H3 and H10 H12 o Ifusing a Hellma plate Pipette 200 pL of buffer into the wells surrounding the 3 3 nM wells CBUF in the grid For the Sensitivity
31. Microplate slide holder Isolation table Objective adapter collar wrench Objective setup plate 3 32 hex wrench Models with an external dispense module packed separately with the following accessories FireWire laptop card and power supply 8040541 7082121 7083000 Injector Inlet tubes 2 from supply bottles to syringe drives 250 uL syringes 2 BioTek Instruments Inc 1 Unpack and Inspect the Reader 3 Mem art Supply bottles 2 30 mL 7122609 Supply bottle holders 2 8042193 Injector tip cleaning stylus and plastic storage bag 2872304 Strap reagent racks 6 7212035 Models with the gas controller G models packed separately Gas controller unit CO gt 0 control Shipping accessories CO2 0 control Gas Controller Unit CO only Shipping accessories CO only 1 Unpack and Inspect the Reader The Cytation 5 should be removed from the box by two people The instrument with all available modules weighs up to 80 pounds 36 6 kg Save all packaging materials If you need to ship the reader to BioTek for repair or replacement you must use the original materials Using other forms of commercially available packaging or failing to follow the repackaging instructions may void your warranty During the unpacking process inspect the packaging reader and accessories for shipping damage If the reader is damaged notify the carrier and your BioTek representative Keep the shipping boxes and the p
32. Monochromator Based Fluorescence System To pass the Detection Limit ul ALE must be less than or equal to EX 485 nm EM 528 nm Top Bottom 20 pM Linearity Test 1 Calculate the Mean of the four wells for each concentration in columns 1 5 2 Perform linear regression using these values as inputs Filter and Monochromator Based Fluorescence System y Mean of the 1000 pM wells Mean of the 250 pM wells Mean of the 125 pM wells Mean of the 62 5 pM wells Mean of the 500 pM wells 3 Calculate the R Square value it must be gt 0 950 to pass Fluorescence Polarization FP Test 1 Using the raw data from the Parallel read e Calculate the Mean Blank wells A6 H6 e Calculate the Signal for each HPR well Subtract the Mean Blank from its measurement value e Calculate the Signal for each LPR well Subtract the Mean Blank from its measurement value 2 Using the raw data from the Perpendicular read e Calculate the Mean Blank wells A6 H6 e Calculate the Signal for each HPR well Subtract the Mean Blank from its measurement value e Calculate the Signal for each LPR well Subtract the Mean Blank from its measurement value Cytation 5 76 Instrument Testing 3 Calculate the G Factor for each LPR well Parallel LPR Sign 1 0 02 Perpendicular LPR Signal 1 0 02 Calculate the Mean G Factor 5 Calculate the Polarization value in mP for each HPR well PLPR Parallel HPR Signal Mean
33. Replace the trough in the microplate carrier Clean the Priming Plate Applies only to Cytation 5 models with a dispenser Clean the priming plate regularly to prevent bacteria growth and residue buildup Wash the plate in hot soapy water using a small brush to clean in the corners Rinse thoroughly and allow it to dry completely Clean the Dispense Tubes and Injectors Applies only to Cytation 5 models with a dispenser The Cytation 5 s dispense tubes and injectors require routine cleaning at least quarterly and possibly more frequently depending on the type of fluids dispensed Required Materials Protective gloves Safety glasses Mild detergent Clean lint free cotton cloths Deionized or distilled water Stylus stored in a plastic cylinder affixed to the rear of the dispense module or reader PN 2872304 Remove the Dispense Tubes and Injector Holders injector tip holder BioTek Instruments Inc Clean the Dispense Tubes and Injectors 53 1 Open the door on the front of the reader Grasp the injector tip holder by the tab and pull it up out of its socket 3 Using your fingers remove the thumbscrews securing the light shield to the top of the reader and slide the shield up the outlets tubes 4 Slide the injector tip holder through the hole in the top of the reader 5 Turn each tube s thumbscrew counterclockwise and gently pull each tube from its injector tip 6 On the dispense module turn each ou
34. Table 4x Comer Clips 2 Place the isolation table in the selected installation location 3 Place the instrument on the table as shown next Cytation 5 6 Installation The isolation table contains material that dampens vibration Over time this material becomes compressed and can lose effectiveness The isolation table has a color indicator that turns from green to red to show when the table should be replaced because the dampening material has been compressed gt e ee 5 Remove the Shipping Hardware D Remove all shipping hardware before you turn on the reader 1 Locate the shipping hardware The figures below depict a Cytation 5 with the filter module and imaging module Top Filter Ship Bracket lt BioTek Instruments Inc 6 Install the Power Supply 7 Bottom Filter Slide ship bracket Objective ship l Y bracket 2 Open the top door and remove the reusable zip tie and desiccant packet from the desiccant anchor 3 If equipped use the supplied screwdriver to remove the top filter shipping bracket 4 Open the front door and using the supplied screwdriver remove the carrier shipping bracket 5 If equipped with the imaging module use a 9 64 hex wrench to remove the bottom filter slide ship bracket 6 Push the filter slide back and remove the two piece objective ship bracket 7 Store the shipping hardware in a safe location in case the instrument
35. Type setting in each Gen5 protocol should match the plate you are actually using Cytation 5_FI_T_SF prt This procedure contains two Read steps using filters to test the top optics one for the Corners Test and one for the Sensitivity Linearity Test Parameter Default Setting Plate Type Costar 96 black opaque 3915 Read Step 1 Read Type Endpoint Run Time 00 00 45 Kinetic Loop Interval 00 00 03 Reads 16 Normal Delay after plate movement 350 msec Read Speed Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Run Time 00 01 35 Kinetic Loop Interval 00 00 06 Reads 16 Step Label Sensitivity Read Buffer Read Wells C9 E9 Filter Set 1 Green BioTek Instruments Inc Fluorescence Liquid Tests 79 EX 485 20 nm EM 528 20 nm Optics Position Top 510 nm Gain Auto Use first filter set gain from FIRST Read Step Normal Delay after plate movement 350 msec Read Speed Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Read Height Read Step 3 Detection Method Read Type 7 00 mm Fluorescence Endpoint Corners Read A1 A3 A10 A12 H1 H3 H10 H12 1 Green EX 485 20 nm EM 528 20 nm Top 510 nm Auto Scale to High Wells A3 30000 Read Wells Filter Set Filters Optics Position Normal Delay after plate movement 350 msec Read Speed Measurements per data point 50 Lamp Energy Low Dynamic Range Standard 7
36. a prt or Cytation 5 F LumTest_Glowell prt protocols described in this section create the filter set shown below Filter Set 1 Filter SetName Open i Excitation Plug Mirror lt none gt w none gt Ex Min Max Em Min Max EA mn Emission Hole w Harta Plate Test Materials e Harta Luminometer Reference Microplate PN 8030015 e Harta Plate Adapter PN 1222205 e Filter cube PN 8040553 LUM Filter Block or a filter cube with a plug in EX position 1 or 2 and a hole in the corresponding EM position e Gen5 protocol described started on page 88 BioTek Instruments Inc Luminescence Test 85 Procedure 1 Turn on the Harta reference plate using the I O switch on the back of the plate 2 Check the plate s battery by pressing the test button on the back of the plate and ensuring that the test light turns on The test light may be difficult to see in bright light Change your angle of view or move to a darker environment if you cannot see it 3 Place the Harta plate adapter on the reader s carrier then place the test plate on top of the adapter 4 Create an experiment based on the Cytation 5 F LumTest_Harta prt or Cytation 5 M LumTest_Harta prt protocol and read the plate 5 Calculate and evaluate the results as described on page 86 Be sure to turn off the Harta plate when you are finished with the test to preserve battery life Plate Layout Cytation 5 F LumTest_Harta prt battery ba
37. ace Place all packaging materials into the shipping box for reuse if the joystick needs to be shipped 3 Locate the joystick cable Plug one end into the port on the back of the joystick Plug the other end into the joystick port on the rear of the reader 9 Install the Dispenser Place the dispense module on top of the reader or on top of the gas controller if equipped Do not place the dispenser next to the reader Syringe Inlet tubes Outlet tubes _ Injector tip Light shield a 4 BioTek Instruments Inc 9 Install the Dispenser 9 Open the plastic bag containing the injector tube and tips Remove the clear plastic shrouds from the tubes Remove the two inlet tubes from their plastic canisters Identify the two syringe valves on the dispense module Each is labeled with a left pointing arrow When installing the inlet and outlet tubes do not use any tools Finger tighten only 4 Screw the fitting of one inlet tube into the right side of the Syringe 1 valve 5 Identify the 1 outlet tube and screw it into the left side of the Syringe 1 valve 6 Repeat these steps to attach the inlet and outlet tubing for Syringe 2 It is critical that the tubing is installed in the correct ports Otherwise injected fluid may miss the intended well Remove the round tubing feed through cover from the top of the reader 2 screws Store the cover and screws with the shipping hardware in case the reader needs
38. aceable Sodium Borate Reference Standard pH 9 18 e g Fisher Scientific 1 L Sodium Borate Mfr 159532 or equivalent or Phosphate Buffered Saline PBS pH 7 2 7 6 e g Sigma tablets Mfr P4417 or equivalent and pH meter or pH indicator strips with pH range 4 to 10 e Sodium Fluorescein Powder 1 mg vial BioTek PN 98155 BioTek Instruments Inc Fluorescence Liquid Tests 69 If testing both Top and Bottom optics mono based fluorescence only A new clean 96 well glass bottom Greiner SensoPlate Mfr 655892 or a clean Hellma Quartz 96 well titration plate Mfr 730 009 QG or equivalent If testing the Top optics only A new clean 96 well solid black microplate such as Corning Costar 3915 or equivalent Excitation filter 485 20 nm installed Emission filter 528 20 nm installed 510 nm dichroic mirror installed Fluorescence Polarization FP Test e Anew clean 96 well solid black microplate such as Corning Costar 3915 A Greiner SensoPlate can also be used The FP Test can be performed in conjunction with the top Corners Sensitivity Linearity Tests in the same microplate e The recommended test solutions are available from Invitrogen Corporation in their FP One Step Reference Kit PN P3088 or from BioTek PN 7160014 This kit includes Green Polarization Reference Buffer 15 mL Green Low Polarization Reference 4 mL Green High Polarization Reference 4 mL The kit also includes
39. ackaging materials for the carrier s inspection BioTek will arrange for repair or replacement immediately 1 Open the shipping box remove the instrument from the box and place it on a level stable surface 2 Place the packaging materials back into the shipping box for reuse if the instrument needs to be shipped again 3 For the instruments with the imaging module Open the accessories box and remove the isolation table Cytation 5 4 Installation 2 Unpack and Inspect the Dispenser If applicable 1 Open the shipping box Remove the accessories box and foam insert that contains the injector tubing and bottle holders Lift out the dispenser and place it on a level surface Open the accessories box and remove its contents Place all packaging materials into the shipping box for reuse if the dispenser needs to be shipped 3 Unpack and Inspect the Gas Controller If applicable 1 Open the shipping box Remove the accessories and set them aside Lift out the gas controller and place it on a level surface Place all packaging materials into the shipping box for reuse if the gas controller needs to be shipped 4 Select an Appropriate Location Install the reader on a level stable surface in an area where ambient temperatures between 18 C 64 F and 30 C 86 F can be maintained Leave at least six inches of space between the instrument s rear panel and any other object This space ensures proper air
40. ader Depending on the model Cytation 5 detection modes include fluorescence intensity FI fluorescence polarization FP time resolved fluorescence TRF luminescence UV visible absorbance imaging and alpha The instrument is modular and upgrade options are available contact BioTek Customer Care for more information The reader is computer controlled using Gen5 software for all operations including data reduction and analysis The Cytation 5 is robot accessible and compatible with the BioStack 3 and BioStack 4 Gen5 supports OLE automation to facilitate the Cytation 5 s integration into an automated system Refer to the Gen5 Help system for operational and data analysis instructions The Cytation 5 Operator s Manual contains recommendations for ensuring optimum performance External Components 1 The entry for the dispense outlet tubes and injectors if equipped 2 Filter cube access door 3 The microplate carrier eject button 4 The light blocking microplate carrier access door 5 The power switch 6 LED cubes imaging filter cubes and objectives access door BioTek Instruments Inc Internal Components 27 Dispenser port Gas controller hookup 1 2 Joystick port 3 USB port 4 Power inlet 5 6 FireWire port Internal Components The filter cube can contain excitation and emission filters mirrors and polarizing filters Preconfigured cubes are available from BioTek Filter Cube or you can
41. air bubbles can get trapped in the system and affect injection volumes Air bubbles in the system can also result in fluid spraying or scattering inside the reader Both types of primes require a fluid reservoir to be present on the microplate carrier e The priming plate is placed on the microplate carrier for a Prime operation to prime the dispense system with fluid e The tip priming trough is placed in the rear pocket of the carrier and is used for performing the Tip Prime before dispensing The trough holds up to 1 5 mL of liquid and must be periodically emptied and cleaned by the user Do not perform tip priming when using tall plates Generally plates with fewer than 96 wells are too tall for error free tip priming and tip priming is rarely required for these larger volume plates The priming tray should be empty before priming and contain fluid after priming Imaging System Instruments with the imaging module can perform image reads view and capture images in manual mode and save the images for later analysis The imaging module comprises up to four LED cubes and four imaging filter cubes up to six objectives and a CCD digital camera which captures images directly through the selected objective and filter cube assembly The imaging module supports two modes manual mode and experiment mode BioTek Instruments Inc Internal Components 31 Camera Gen controls the CCD camera via FireWire Using Gen5 you can focu
42. all off Cytation 5 14 Installation 5 Insert the FireWire Express card with the label facing up into the appropriate slot on your laptop 6 Plug the power cord into the card then plug the cord into a power outlet FireWire cable to reader Install the FireWire Driver You must install the PCI Express card before performing this step 1 Navigate to the Gen5 program files on your computer for example CA Program Files BioTek Gen5 2 07 BioTek Instruments Inc 14 Install the Imager Module 15 2 Open the Firewire Drivers folder and then open the folder appropriate for your computer Windows_32 for Windows 32 bit or Windows_64 for Windows 64 bit 3 In Windows 7 and higher right click InstallPGRDriver bat and select Run as Administrator to run the driver installer When the installer is finished a message appears SUCCESS Installed package lt path to package gt If you do not see this message contact BioTek TAC 4 After installing the FireWire driver restart your computer 5 When the reboot process is complete insert one end of the FireWire cable into the back of the reader and insert the other end into the new port in the card you installed on your computer Establish Communication with the Camera 1 From the Gen5 main screen select System gt Instrument Configuration select Cytation 5 and then click View Modify 2 Click Camera Information If communication is successful Gen5 di
43. alternate test solutions listed in the next section e 100 mL graduated cylinder and 10 mL pipettes if not using BioTek s Green Test Dye Solution e Gen software installed on the host PC e Gen protocols described on page 98 Cytation 5 96 Instrument Testing Alternate Test Solutions 80 uL of test solution with 150 uL of deionized water should read between 1 300 and 1 700 OD at 405 750 nm The solutions should be at room temperature If you do not have BioTek s Green Test Dye Solution PN 7773003 prepare a dye solution using one of the following methods Using BioTek s Blue and Yellow Concentrate Dye Solutions Ingredient Quantity Concentrate Blue Dye Solution PN 4 0 mL 7773001 125 mL QC Yellow Solution PN 7120782 125 mL Using FD amp C Blue and Yellow Dye Powder Ingredient Quantity FD amp C Blue No 1 0 200 grams FD amp C Yellow No 5 0 092 grams Sodium Azide N3Na 0 100 gram Procedure The Cytation 5 Operator s Manual contains the procedure for models without absorbance capabilities 1 Ifyou have not already done so create the Gen5 protocols Cytation 5 Disp 1 Test prt and Cytation 5 Disp 2 Test prt starting on page 98 2 Prime both dispensers with 4000 uL of deionized or distilled water 3 Purge both dispensers with the Volume set to 2000 uL This prevents the water from diluting the dye Remove the inlet tubes from the supply bottles 4 Filla beaker with at least 20 mL of the
44. arrier Shake Definitions at 59 Maximum Shaking Amplitude Based on Assay Volume and Plate Type e e e elre 0O N dODd UIA a WO NINININI N NINI NI NIN OII NI AIAJ BR WIN F UJ O If the wells of a microplate are almost full vigorous shaking can result in spillage inside the instrument The following table is designed to help avoid this issue Find your microplate type and sample volume to ascertain the acceptable shake amplitude on your instrument Sample nar Orbital Orbital Double Orbital Double Orbital Volume Slow Fast Slow Fast 6 well Microplate 0 3 mL 1 6 mm 1 6 mm 1 6 mm 1 6 mm mm 1 6 mm Cytation 5 34 Getting Started Sample near Orbital Orbital Double Orbital Double Orbital Volume Slow Fast Slow Fast 57m imm remm amm jimm no 3mm No 12 well Microplate 24 well Microplate mm 48 well Microplate 0 0 5 mL 1 6 mm mm 96 well Microplate mm Gen5 Software BioTek Gen5 software supports all Cytation 5 reader models Use Gen5 to control the reader the imaging module if equipped the dispense module if equipped and the stacker if equipped perform data reduction and analysis on the measurement values print or export results and more This section provides brief instructions for working with Gen to create protocols and experiments and read plates Refer to the Gen5 Help system for more information and to learn about the various license levels available BioTek Instrumen
45. as a result Filters should be inspected and cleaned at least every three months You ll need Isopropyl ethyl or methyl alcohol 100 pure cotton balls or high quality lens cleaning tissue Cloth gloves e Magnifying glass BioTek Instruments Inc Clean the Objectives 49 Do not touch the filters with your bare fingers 1 Open the access door on the front of the instrument Slide the filter cube out of its compartment 2 Inspect the glass filters for speckled surfaces or a halo effect This may indicate deterioration due to moisture exposure over a long period of time If you have any concerns about the quality of the filters contact your BioTek representative 3 Using cotton balls or lens cleaning tissue moistened with a small amount of high quality alcohol clean each filter by lightly stroking its surface in one direction 4 Use a magnifying glass to inspect the surface remove any loose threads left from the cotton ball 5 Replace the filter cube and close the door Clean the Objectives Applies only to models with the imaging module The objectives used in the Cytation 5 should be cleaned when necessary using optical grade swabs or lens paper moistened with lens cleaning solution or deionized water Do not rub the lens Materials e Air puffer e Tweezers e Magnifying glass e Lens cleaning tissue e Optical grade swabs e Cleaning solvent Recommended Cleaning Solvents AOT Eee Nine a ee e Solve
46. ass a standard interference filter with a defined central wavelength and bandwidth e Long Pass cutoff filters that transmit longer wavelengths and block shorter wavelengths e Short Pass cutoff filters that transmit shorter wavelengths and block longer wavelengths e Select PLUG to indicate the presence of a plug e Select HOLE to indicate an empty location Configuring the System for Luminescence Measurements If your tests require that light emitted from the samples remain unfiltered the Emission filter position in the filter cube should be empty 1 Click System gt Optics Library gt Filter Cubes 2 Select the checkbox in the On Site column for the filter cubes you have available Injector System The tubing and injectors should be cleaned at least every three months Inspect the injector system daily for leaks preferably immediately after priming and whenever tubing changes have been made BioTek Instruments Inc Internal Components 29 If a syringe is leaking it may need to be replaced Dispense Module The dispense module sits on top of the reader or gas controller and pumps fluid from the reagent bottles to injector located inside the instrument Fluid is injected into one well at a time The injectors support plate types from 6 to 384 well plates 1 Two 250 uL syringes draw fluid from the supply bottles 2 Inlet tubes transport fluid from the supply bottles to the syringes These tubes are short
47. ch gives the limit of detection at a signal to noise ratio of one where noise is defined as three times the standard deviation of the background wells Monochromator Based Fluorescence 250 700 nm with low noise PMT Excitation range 250 900 nm with red shifted PMT 250 700 nm with low noise PMT 300 700 nm for emission scans with low noise PMT Emission range 250 900 nm with red shifted PMT 300 900 nm for emission scans with red shifted PMT Variable from 9 nm to 50 nm in 1 nm increments both Bandpass ee _ excitation and emission IO Unete lt 20 seconds sweep mode 96 well microplate interval Plate In Plate Out Speed lt 35 seconds sweep mode 96 well microplate Sensitivity Sodium Fluorescein in phosphate buffered saline PBS DL lt 20 pM top or bottom read 5 pM typical Excitation 485 nm Emission 528 nm Methylumbelliferone MUB in carbonate bicarbonate buffer CBB DL lt 0 16 ng mL 0 91 nM top read Excitation 360 nm Emission 460 nm Propidium Iodide PI in PBS DL lt 62 5 ng mL bottom read Excitation 485 nm Emission 645 nm Cytation 5 108 Specifications Fluorescence Specifications Filter Based The Cytation 5 measures fluorescence with filters from the top of 6 to 384 well plates Plate In Plate Out Speed lt 35 seconds for filter set sweep mode 96 well microplate Fluorescence Intensity DL lt 10 pM 3 pM typical solution of Sodium Fluorescein in PBS Excitation 485 20
48. col described starting on page 61 3 Create a new experiment in Gen using the AF_Reliability_ lt x gt prt protocol with lt x gt representing the objective you are using for each test 4 Click Plate gt Read Plate save the experiment then click OK 5 When the read is finished analyze the results as described below Analyze the Results 1 For the set of 100 data points in well F7 calculate the Mean Slope and Intercept If using Microsoft Excel use the SLOPE and INTERCEPT functions 2 For each data point calculate the Residual value Slope data point sequence Intercept Example 325 0 1 325 9 0 9 Residual for data point 1 3 For each data point compare the absolute value of the Residual with the objective s Limit um value in the chart below e g 95 4 for 2 5X If the absolute value of the Residual is greater than the Limit note 1 for that data point otherwise note 0 Depth of Field Objective Magnification NA e um A nm DOF pm Limit um 10X 10 0 3 6 45 377 6 3 12 7 4 After conducting this comparison for all 100 data points add up the notations and divide the sum by 100 this is the Measured Autofocus Failure Rate The Measured Autofocus Failure Rate must be lt 1 0 to Pass 5 In the Gen plate matrix select the FM Ratio 0 DAPI 377 447 data set Refer to the chart below for passing criteria for the value displayed in well FZ Objective FM Ratio must be BioTe
49. curacy The Absorbance Plate Test compares the reader s optical density and wavelength measurements to NIST traceable values The Absorbance OD Standards section contains NIST traceable standard OD values for the filters at several different wavelengths We recommend testing at six wavelengths those at or close to the wavelengths used in your assays Setup 1 Obtain the certificates that came with the Test Plate 2 Start Gen5 and from the main screen select System gt Diagnostics gt Test Plates gt Add Modify Plates BioTek Instruments Inc Absorbance Plate Test 57 Click Add The Absorbance Test Plate dialog appears Select the appropriate Plate Type and enter the plate s serial number Enter the Last Certification and Next Certification dates from the calibration sticker on the Test Plate If the wavelength values in the top row of the grid in Gen5 are appropriate for your tests enter the OD values from the data sheet into the grid Make sure you enter the correct value for each well wavelength combination Select the number of Peak Wavelength tests to run up to 4 based on the number of expected peak wavelength values provided on the certificate Enter the Expected Peak value s from the certificate and set the Test Range and values e If the C6 filter is Holmium or Erbium glass the certificate contains two Spectral Band Pass tables The Cytation 5 has a band pass wider than 5 nm for wavelengths greater tha
50. cy Alpha Jig PN 7162512 Rev F or higher The alpha laser has been factory calibrated to meet specification BioTek Instruments Inc has developed a test protocol that can be used with Alphascreen Omnibeads to verify the functionality of the alpha laser system Because the detector for the alpha system is functionally and optically identical to the luminescence system the luminescence test may be used to verify detector functionality The Crosstalk test is a measure of how well the optical system can distinguish the signals emitted from the well being read from those of any adjacent well This test also determines the signal to noise ratio SNR of the test plate and verifies that the signal is at an acceptable level for the sample material used The test is designed for use with Alphascreen Omnibeads and it is assumed that 96 well plates are used with 100 uL well volumes Required Materials e Recommended test solution Alphascreen Omnibead Assay kit available from PerkinElmer PN 6760626 e Buffer Phosphate Buffered Saline PBS pH 7 2 7 6 e g Sigma tablets Mfg P4417 or equivalent e Clean 96 well solid white microplate e 15 mL conical bottom polypropylene sample tube e Alpha filter cube e Gen5 protocols described on page 94 Test Solutions Alphascreen Omnibeads are light sensitive All tests should be performed under subdued laboratory lighting of less than 100 lux 1 Prepare the PBS buffer solution a Optiona
51. e to find your samples then click Save settings Gen5 imports your exposure settings Cytation 5 40 Getting Started and the values for Horizontal and Vertical offset from the center of the well to your read step 4 Select Auto to turn Auto Exposure back on Gen5 will auto expose your image retaining the well offset imported from manual mode You can also enter the values for Horizontal and Vertical offset from center of well if you had determined them during previous testing Dispense Module Control This section applies only to models with injectors Gen5 is used to perform several dispense functions such as initialize dispense prime and purge The Prime and Purge functions are introduced here See the Gen5 Help system for more information Priming and purging routines are used to clean the fluid paths Prime Before running an experiment with a dispense step prime the system with the fluid to be used 1 Place the priming plate on the carrier Fill the supply bottle with a sufficient volume of the fluid to be used for the prime and the assay Insert the appropriate inlet tube into the bottle 3 In Gen select System gt Instrument Control gt Cytation 5 and click the Prime tab 4 Select the Dispenser number 1 or 2 associated with the supply bottle 5 Enter the Volume to be used for the prime The minimum recommended prime volume is 2000 uL 6 Select a prime Rate in uL second 7 Click Prime to sta
52. e is no such label the instrument may be used only for research and development or other non clinical purposes Quality Control It is considered good laboratory practice to run laboratory samples according to instructions and specific recommendations included in the assay package insert for the test to be conducted Failure to conduct Quality Control checks could result in erroneous test data BioTek Instruments Inc Warnings v Warnings AS Operate the instrument on a level stable surface away from excessive humidity Bright sunlight or strong incandescent light can reduce the linear performance range of the instrument Measurement values may be affected by extraneous particles such as dust in the microplate wells A clean work area is necessary to ensure accurate readings When operated in a safe environment according to the instructions in this document there are no known hazards associated with the instrument However the operator should be aware of certain situations that could result in serious injury these may vary depending on the instrument model See Hazards and Precautions Hazards The following hazards are provided to help avoid injury Warning Power Rating The instrument s power supply or power cord must be connected to a power receptacle that provides voltage and current within the specified rating for the system Use of an incompatible power receptacle may produce electrical shock and fire
53. e the Maintenance chapter for decontamination instructions Remove the microplate and tip prime trough if equipped from the carrier before shipment Spilled fluids can contaminate the optics and damage the instrument The instrument with all available modules weighs up to 80 Ibs 36 3 kg Use two people when lifting and carrying the instrument The instrument s packaging design is subject to change If the instructions in this section do not appear to apply to the packaging materials you are using please contact BioTek s Technical Assistance Center for guidance Replace the shipping hardware before repackaging the reader Please contact BioTek if you have misplaced any of these items Carrier ship bracket PN 1220510 Carrier ship bracket screws PN 1032190 Top filter shipping bracket PN 8042187 Bottom filter slide ship bracket PN 1380501 Objective ship bracket PN 1380503 If you need to ship the Cytation 5 and or the dispense module to BioTek for service or repair be sure to use the original packaging materials Other forms of commercially available packaging are not recommended and can void the warranty The shipping materials are designed to be used no more than five times If the original materials have been damaged lost or used more than five times contact BioTek to order replacements BioTek Instruments Inc Getting Started 26 Getting Started Overview The Cytation 5 is a hybrid multi mode microplate re
54. ease virtual memory using the following instructions to prevent errors 1 From the Windows Start menu go to Control Panel and select System 2 Inthe left pane select Advanced system settings 3 Inthe System Properties dialog on the Advanced tab in the Performance area click Settings 4 Inthe Performance Options dialog on the Advanced tab in the Virtual Memory area click Change 5 Clear Automatically manage paging file size for all drives if it is selected 6 Select Custom Size enter the following minimum and maximum values and then click OK e Initial size 10 GB e Maximum size 20 GB Cytation 5 32 Getting Started You may need to restart your computer for the change to take effect Shaking System Three shake modes are available for selection in Gen5 Linear Orbital and Double Orbital A linear shake simply moves the carrier s y axis back and forth in a line An orbital shake moves both carrier axes to scribe a circle With double orbital the carrier scribes a figure eight pattern For any mode a slider bar in the software allows you to adjust the shake frequency from Slower to Faster With each adjustment the corresponding cycles per minute CPM is displayed For either orbital mode you can further expand the frequency options by clicking a Slow or Fast button Shake Step Duration MM155 Continuous Shake Slower a Slow Fast BioTek Instruments Inc Shaking System 33 C
55. econd shake the 80 uL read at 405 750 nm and the 20 and 5 uL read at 630 750 nm When processing is complete save the file with an identifying file name Remove the plate from the carrier and set it aside Repeat steps 5 11 using Cytation 5 Disp 2 Test prt See the instructions for analyzing the results below When all tests are complete prime both dispensers with at least 5000 uL of deionized water to flush out the green dye solution Results Analysis The pass fail criteria for each set of 32 wells with the same dispense volume is based on the calculated coefficient of variation CV and Accuracy Error For each volume dispensed 80 uL 20 uL 5 uL for each dispenser 1 2 Calculate the Standard Deviation of the 32 wells Calculate the Mean of the 32 wells Calculate the CV Standard Deviation Mean x 100 Calculate the Accuracy Error Actual Weight Expected Weight Expected Weight 100 Cytation 5 98 Instrument Testing Expected Weights for 32 wells 80 uL 2 560 g 20 uL 0 640 g 5 uL 0 160 g It is assumed that one gram is equal to one milliliter 0 Dispense Volume To pass oCV must be SS Error must be Gen5 Test Protocols for Models with Absorbance Capabilities Perform the steps in the following sections to create two protocols for testing Dispensers 1 and 2 When finished save the files as Cytation 5 Disp 1 and 2 Test prt Define the Procedure The protocol
56. eguards specified in this manual could result in a hazardous condition Warning Software Quality Control The operator must follow the manufacturer s assay package insert when modifying software parameters and establishing reading methods Failure to conduct quality control checks could result in erroneous test data Warning Reader Data Reduction Protocol No limits are applied to the raw measurement data All information exported via computer control must be thoroughly analyzed by the operator Warning Internal Voltage Always turn off the power switch and unplug the power supply before cleaning the outer surface of the instrument or removing its top case Warning Potential Biohazards Some assays or specimens may pose a biohazard This hazard is noted by the symbol shown here Adequate safety precautions should be taken as outlined in the assay s package insert Always wear safety glasses and appropriate protective equipment such as chemical resistant rubber gloves and apron Warning LED Lights Serious eye injury may occur if you stare directly at the LED during operation of the light This hazard is noted by the symbol shown here Warning Pinch Hazard Some areas of the dispense module can present pinch hazards when the instrument is operating The module is marked with the symbol shown here Keep hands fingers clear of these areas when the instrument is operating Precautions The following precautions are provided to he
57. ess is complete carefully remove the priming plate from the carrier and empty it 7 Repeat the process for Dispenser 2 Leave the water in the system overnight or until the instrument will be used again Purge the fluid from the system see below and then prime with the dispense reagent before running an assay To purge the fluid from the system Place the inlet tubes in empty supply bottles or a beaker Select System gt Instrument Control gt Cytation 5 Click the Prime tab and select Dispenser 1 Set the Volume to 2000 UL Click Purge to start the process oS oS S hy When the purge is complete repeat the process for Dispenser 2 After purging the system you may wish to run a quick Dispense protocol to visually verify the dispense accuracy Empty Clean the Tip Priming Trough Applies only to Cytation 5 models with a dispenser The tip priming trough is a removable cup located in the rear pocket of the microplate carrier used for performing the Tip Prime The trough holds about 1 5 mL of liquid and must be periodically emptied and cleaned by the user Gen5 will instruct you to do this at the start of an experiment that requires dispensing 1 Extend the microplate carrier and carefully remove the tip priming trough from the carrier Cytation 5 52 Maintenance 2 Wash the trough in hot soapy water Use a small brush to clean in the corners 3 Rinse the trough thoroughly and allow it to dry completely 4
58. figuration area select the objective for the position or positions you want to define or select None if you are removing an objective from the instrument Click Access 4 Inthe LED and Imaging Filter Cube Configuration area select the filter cube for the position you want to define or select None if you are removing the filter cube from the instrument The corresponding LED cube part number is filled in automatically 5 Click Send Values 6 For LED cube and imaging filter cube changes follow the on screen procedure 3b Install the Objectives Before installing a 20X 40X or 60X objective either phase or standard set its correction collar to match your plate type See the Cytation 5 Operator s Manual for more information and instructions After defining the objectives and their locations in Gen 1 Turn the knob on the side access door to release the latch and open the door Cytation 5 18 Installation If you are installing an objective with a correction collar e a 20X 40X or 60X objective be sure to grasp the objective by the adapter not by the correction collar to avoid changing the correction collar settings 2 Screw each objective into its defined position Do not overtighten the objectives 3c Install the LED Cubes and Imaging Filter Cubes 1 With the instrument powered off slide the filter slide out of the instrument 2 Place the new LED cube in the appropriate position Position 1 2 3 or
59. file an identifying name These instructions briefly describe how to create an experiment and then read a plate in Gen5 See the Gen5 Help system for complete instructions Open a new experiment Select the desired protocol and click OK Select a plate in the menu tree and read it SR When the read is complete measurement values appear in Gen Select the desired data set from the Data list 5 Select File gt Save and give the file an identifying name Imaging Module Manual Mode Applies only to models with the imaging module The following sections briefly describe how to use the Gen5 imaging module in manual mode See the Gen5 Help for more complete instructions and descriptions of these features Focusing the Camera 1 From the Task Manager select Read Now gt Run Imager Manual Mode KA Alternatively from the main Gen screen click 2 Click Find Image Gen5 automatically sets the exposure and focuses on the image If the displayed image requires additional fine tuning e Click Auto Expose or manually adjust the Exposure settings until you see an image BioTek Instruments Inc Gen5 Software 37 e Click Auto Focus or click the arrow buttons until the image is in focus e Repeat these two steps until the image is exposed and focused to your liking Capture and Save an Image When you have a satisfactory image you can capture and save it for later analysis 1 When an image in the Capture dialog is in foc
60. green dye solution Prime both dispensers with 2000 uL of the solution When finished remove the priming plate from the carrier 5 In Gen5 create an experiment based on Cytation 5 Disp 1 Test prt BioTek Instruments Inc Dispense Module Tests 97 6 Place a new 96 well microplate on the balance and tare the balance 7 Place the plate on the microplate carrier Running a dispense procedure without placing a plate in the reader will result in contamination of the reader from spilled liquid 10 11 12 Lo 14 When each dispense step is finished you will weigh the plate record the weight tare the balance with the plate on it and then place the plate back on the carrier for the next step MI 0 po SF p Select Plate gt Read and click READ Gen5 prompts you to empty the tip priming trough When ready click OK at the Load Plate dialog to begin the experiment The sequence is as follows Dispense 80 uL well to columns 1 4 Remove the plate and weigh it Record the weight and tare the balance Place the plate on the carrier and dispense 20 uL well to columns 5 8 Remove the plate and weigh it Record the weight and tare the balance Place the plate on the carrier and dispense 5 uL well to columns 9 12 Remove the plate and weigh it Record the weight Manually pipette 150 uL of deionized or distilled water into all 12 columns on top of the green test dye solution Place the plate on the carrier for a 15 s
61. iant Flux 1 0 000333 number of days since calibration 4 Convert the Corrected Radiant Flux value to attomoles Corrected Radiant Flux 0 021 1800 Cytation 5 88 Instrument Testing 5 Calculate an error factor for the Corrected Radiant Flux amol Corrected Radiant Flux in amol Measurement Uncertainty 100 6 Calculate the min max criteria for the Corrected Radiant Flux amol MIN Corrected Radiant Flux in amol Error Factor MAX Corrected Radiant Flux in amol Error Factor 7 Calculate the Signal to Noise Ratio Measurement value of the Glowell Mean of Column 9 3 x Standard Deviation of Column 9 8 Calculate the Detection Limit Corrected Radiant Flux in amol Signal to Noise Ratio 9 Calculate the min max criteria for the Detection Limit MIN MIN for Corrected Radiant Flux in amol Signal to Noise Ratio MAX MAX for Corrected Radiant Flux in amol Signal to Noise Ratio If the reader is equipped with the low noise PMT the detection limit must be lt 75 amol to pass If the reader is equipped with the red shifted PMT the detection limit must be lt 500 amol to pass Gen5 Protocol Reading Parameters Cytation 5 F LumTest_Harta prt Read Wells Filter Sets Excitation Emission Delay after plate movement BioTek Instruments Inc Luminescence Test 89 Delay after plate movement e Delay after plate movement Cytation 5 M LumTest_Harta prt Plate Type 8030015 Harta w o
62. ior to pipetting Alternatively sonicate or vortex the container e Mix 10 uL of FluoSpheres with 10 mL of deionized water in a 15 mL conical bottom polypropylene sample tube This yields a 20 nM equivalent suspension e Shake the vial vigorously for 30 seconds prior to pipetting Alternatively sonicate or vortex the container e Mix 10 uL of 20 nM suspension with 10 mL of deionized water in a 15 mL conical bottom polypropylene sample tube This yields a 20 pM equivalent suspension e Refrigerate any unused portions of the FluoSpheres The temperature must be between 2 C to 6 C The prepared TRF plate can be kept for a maximum of seven days if covered and stored in the dark between 2 C to 6 C Allow the plate to sit at room temperature for approximately 15 minutes prior to use Procedure 1 If you have not already done so create the Gen5 protocols described starting on page 77 2 Ifyou have not already done so prepare the solutions for the tests you plan to perform See Fluorescence Test Solutions on page 70 Refer to the pipette maps next for the remaining steps 3 Perform the Corners Sensitivity Linearity tests using the Top optics of the filter based fluorescence system e Pipette the test solutions into a clean 96 well microplate e Create an experiment based on the Cytation 5_FI_T_SF prt protocol Read the plate and then save the experiment Cytation 5 72 Instrument Testing 4 To test
63. ished the instrument is ready to use Troubleshooting Auto Calibration If error messages indicate a problem with the imaging Auto Calibration process 1 In Gen5 click System gt Instrument Configuration gt Cytation 5 gt View Modify gt Setup BioTek Instruments Inc 3 4 15 Configure the Dispensers in Gen5 21 On the Imaging Configuration tab set the value of all objectives to none and then click Send Values Set the correct values for all objectives and then click Send Values Click Auto Calibration If problems persist contact BioTek TAC 15 Configure the Dispensers in Gen5 Before you use the dispenser with a BioTek reader you must set the calibration values in Gen5 1 In Gen5 go to System gt Instrument Configuration select your instrument and click View Modify Click Setup and then select the Dispenser 1 tab On the keyboard press CTRL SHIFT M to enter maintenance mode for the Dispenser 1 window Enter the syringe calibration values from the label on the rear of the dispenser box See Install the Dispenser Click Send Volumes and then click Get Volumes to verify that the entered values were sent to the instrument Select the Dispenser 2 tab and repeat steps 3 through 5 for Dispenser 2 Cytation 5 22 Installation Dispenser 2 calibration values 16 Test the Dispenser 1 If necessary press the carrier eject button to extend the microplate carrier Place the ti
64. ital apparatus does not exceed Class A limits for radio emissions from digital apparatus set out in the Radio Interference Regulations of the Canadians Department of Communications Le present appareil numerique n emet pas du bruits radioelectriques depassant les limites applicables aux appareils numerique de la Class A prescrites dans le Reglement sur le brouillage radioelectrique edicte par le ministere des Communications du Canada Cytation 5 x Preface User Safety This device has been type tested by an independent laboratory and found to meet the requirements of the following e Underwriters Laboratories UL 61010 1 Safety requirements for electrical equipment for measurement control and laboratory use Part 1 General requirements e Canadian Standards Association CAN CSA C22 2 No 61010 1 Safety requirements for electrical equipment for measurement control and laboratory use Part 1 General requirements e EN 61010 Standards see CE Mark starting on page viii BioTek Instruments Inc Safety Symbols Safety Symbols xi Some of the following symbols may appear on the instrument or accessories Y le Cytation 5 Alternating current Courant alternatif Wechselstrom Corriente alterna Corrente alternata Direct current Courant continu Gleichstrom Corriente continua Corrente continua Both direct and alternating current Courant continu et courant alternatif Gleich und Wechselstrom
65. k Instruments Inc Imaging Tests 61 Troubleshooting If any of the imaging tests fail e Ensure the imaging qualification plate is clean You can use canned air to blow any debris from the surface of the plate e Ensure your objectives are clean e If your imaging tests still fail after cleaning the imaging qualification plate and the objectives contact BioTek TAC Imaging Protocols The information in the following tables represents the recommended reading parameters It is possible that your tests will require modifications to some of these parameters such as the Plate Type The Plate Type setting in each Gen5 protocol should match the plate you are actually using Carrier Level Tests Cytation 5 Meiji 2 5X_CarrierLevel prt Cytation 5 Zeiss 2 5X_CarrierLevel prt Cytation 5 4X_CarrierLevel prt Cytation 5 10X_CarrierLevel prt Parameter Default Setting Read Type Endpoint Plate Type 1222520 imaging qualification plate Read Step 1 Process Mode Well Mode Run Time 18 00 Kinetic Step Interval 2 seconds Reads 10 Objective Meiji 2 5X Zeiss 2 5X 4X 10X Read Wells A1 A12 H1 H12 Color Channel Bright Field Cytation 5 62 Instrument Testing LED 5 Exposure Integration time 50 Gain O Focus method Auto focus with optional scan Minimum focus metric ratio 3 Scan distance 600 Auto Focus Options Scan increment 30 Minimum focus delta 8 of capture exposure for focus 75 Offset f
66. l but recommended Using a 0 45 micron filter filter 200 mL of deionized or distilled water b Follow the manufacturer s instructions on the PBS packaging to create 200 mL dissolving the necessary amount of PBS into the filtered water BioTek Instruments Inc Alpha Detection Test 93 c Stir the solution preferably using a stir table until the PBS is completely dissolved d Check the pH it should be between 7 2 and 7 6 at 25 C 2 Prepare the Omnibead suspension a Shake the container of 5 mg mL Omnibead suspension vigorously for 30 seconds prior to pipetting Alternatively sonicate or vortex the container b Mix 20 uL of 5 mg mL Omnibead suspension with 4 98 mL of PBS in a 15 mL conical bottom polypropylene sample tube This yields a 20 ng mL Omnibead suspension c Refrigerate any unused portions of the Omnibeads The temperature must be between 2 C and 6 C Pipette Map PP tt 2 sta ts o6 7 eo tof at a2 BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF 20 20 20 20 ug mL BUF BUF ug mL BUF BUF ug mL BUF BUF ug mL BUF OMB OMB OMB Procedure Crosstalk Test 1 Pipette 100 uL of PBS in wells A1 A12 B1 B3 B4 B6 B7 B9 B10 B12 C1 C12 and G1 H12 see pipette map BUF wells 2 Pipette 100 uL of 20 ug mL Omnibead suspension into wells B2 B5 B8 and B11 see pipette map 20 ng mL OMB wells Allow the plate to sit at room temperature for approximately 15 minutes pri
67. low preventive maintenance protocols may void the warranty See the Maintenance chapter Caution Shipping Hardware The shipping brackets must be removed before operating the instrument They must be reinstalled before shipping the instrument See the Installation chapter Caution Electromagnetic Environment Per IEC 61326 2 6 it is the user s responsibility to ensure that a compatible electromagnetic environment for this instrument is provided and maintained in order that the device will perform as intended Caution Electromagnetic Compatibility Do not use this device in close proximity to sources of strong electromagnetic radiation e g unshielded intentional RF sources because these may interfere with the proper operation viii Preface CE Mark Based on the testing described below and information contained herein this instrument bears the CE mark Refer to the Declaration of Conformity for specific details Directive 2014 30 EU Electromagnetic Compatibility Emissions Class A The system has been type tested by an independent accredited testing laboratory and found to meet the requirements of EN 61326 1 Class A for Radiated Emissions and Line Conducted Emissions Verification of compliance was conducted to the limits and methods of EN 55011 CISPR 11 Class A In a domestic environment it may cause radio interference in which case you may need to mitigate the interference Immunity The system has been ty
68. lp avoid damage to the instrument 4 Caution Service The instrument should be serviced by BioTek authorized service personnel Only qualified technical personnel should perform service procedures on internal components Caution Spare Parts Only approved spare parts should be used for maintenance The use of unapproved spare parts and accessories may result in a loss of warranty and potentially impair instrument performance or cause damage to the instrument Caution Environmental Conditions Do not expose the system to temperature extremes For proper operation ambient temperatures should remain within the range listed in the Specifications chapter Performance may be adversely affected if temperatures fluctuate above or below this range Storage temperature limits are broader BioTek Instruments Inc Cytation 5 vil Caution Sodium Hypochlorite Do not expose any part of the instrument to the recommended diluted sodium hypochlorite solution bleach for more than 20 minutes Prolonged contact may damage the instrument surfaces Be certain to rinse and thoroughly wipe all surfaces Caution Power Supply Use only the power supply shipped with the instrument Operate this power supply within the range of line voltages listed on it Caution Disposal Dispose of the instrument according to Directive 2012 19 EC on waste electrical and electronic equipment WEEE or local ordinances Caution Warranty Failure to fol
69. ly and the red LED on the carrier switch flashes If this occurs press the carrier eject button to stop the beeping If necessary initiate another system test using Gen to try to retrieve an error code from the reader 1 Turn on the reader and launch Genb If your assays use incubation we recommend enabling Temperature Control and allowing the incubator to reach its set point before running the system test To access this feature from the Gen5 main screen select System gt Instrument Control and click the Pre Heating tab 3 Select System gt Diagnostics gt Run System Test 4 When the test is complete a dialog appears requesting additional information Enter your user name and other information if desired and then click OK 5 The results report appears It shows either SYSTEM TEST PASS or SYSTEM TEST FAIL ERROR error code DETECTED 6 Print the report if desired 7 If the test failed look up the error code in the Cytation 5 Operator s Manual to determine its cause If the cause is something you can fix turn off the reader fix the problem and then turn the reader back on and retry the test If the test continues to fail or if the cause is not something you can fix contact BioTek s Technical Assistance Center Absorbance Plate Test This test uses BioTek s Absorbance Test Plate PN 7260522 to confirm mechanical alignment optical density accuracy linearity and repeatability and wavelength ac
70. mputer The Cytation 5 is equipped with a USB port for connection to the host computer Connect the supplied USB cable between the USB port on the back of the reader and an available USB port on the computer 11 Install Gen5 The Cytation 5 is controlled by Gen5 software running on a host computer There is a certain sequence of events that must be followed to ensure that the software is properly installed and configured Please follow the instructions provided in Gen5 Getting Started Guide to install the software 12 Turn on the Reader If you have not already done so turn on the reader The reader s power switch is located on the lower right corner of the front panel The reader performs a system test When the test is completed the reader extends the microplate carrier The carrier eject button located above to the reader s power switch can be used to extend retract the microplate carrier 13 Establish Communications Before performing this step refer to the instructions that shipped with the USB Driver Software on the Gen5 software media to install the necessary drivers 1 Ifnot already done start Gen5 and log in if prompted The default System Administrator password is admin 2 From the Gen5 main screen select System gt Instrument Configuration and click Add Reader 3 Set the Reader Type to Cytation 5 and click OK to continue 4 Select Plug amp Play BioTek Instruments Inc 14 Install the Image
71. n 285 nm and less than 4 nm for 230 285 nm As a results we recommend you use the expected peak values in the 5 0 nm table for your tests For the Erbium glass any peak can be used For the Holmium glass use the expected peak values closest to 242 279 362 417 and 538 nm For example if your certificate looks like the one below you might choose to run the test at four of the five highlighted Expected Peak Test Range combinations 2 4 nm Spectral Band Pass 5 0 nm Spectral Band Pass Expected Peak Test Range 241 278 287 334 360 417 484 537 642 Expected Peak Test Range 643 545 e Ifyour C6 filter is Didymium glass a single peak wavelength value is provided Enter this value and set the Test Range and values so the range displayed in parentheses is 580 to 590 as shown here Cytation 5 58 Instrument Testing Peak wavelength tests 1 a Expected Peak Test Range 556 le 4 S80 to 590 9 Review all the values you entered Click OK to save the data The information you just entered is available on Gen5 each time the Absorbance Plate Test is performed It may need to be modified after the annual recertification of your test plate Procedure 1 From the Gen5 main screen select System gt Diagnostics gt Test Plates gt Run If prompted select the desired Test Plate and click OK 2 When the Absorbance Test Plate Options dialog appears select Perform Peak Wavelength Test if it is n
72. n Geno Select the desired data set from the Data list Using the Slide Holder Manual Mode 1 4 When prompted upon entering manual mode select one of the slide holders as your plate type The slide holder plate map contains two well positions that correspond with the two slide locations on the slide holder Select the well position that contains the slide you want to image By default the carrier moves so that the middle of the selected slide position is below the objective However the sample on your slide may be in a different location on the slide To find the sample Select your lowest power objective Select either Small step or Large step then use the arrow buttons to move the slide around until you find your sample The cross in the slide display to the right of the arrow buttons shows the general position of the image in relation to the slide The back arrow to the left of the slide display shows the direction that the slide enters the read chamber E After you find your sample you can change to a higher power objective if desired or define a Read step in a protocol using the x and y coordinates of your sample which are displayed below the Well button Experiment Mode 1 In an experiment select one of the slide holders as the plate type and create an Image read step E Clear the Auto box to turn off Auto Exposure then click to enter manual mode Follow steps 2 and 3 in the manual mode procedure abov
73. n the formula to wells A1 to H4 7 Click OK to add the transformation to the Data Reduction list 8 Create another Transformation similar to the above with these characteristics e DS1 set to the 20 and 5 pL Read step at 630 nm e DS2 set to the 20 and 5 pL Read step at 750 nm e New Data Set Name resembling Delta OD 20 and 5 ul_Disp lt gt e Formula DS1 DS2 applied to wells A5 to H12 BioTek Instruments Inc Specifications 102 Specifications General Specifications Microplates The Cytation 5 accommodates standard 6 12 24 48 96 and 384 well microplates with 128 x 86 mm geometry 1536 well plates for imaging only Take3 and Take3 Trio Micro Volume Plates microplate slides T25 cell culture flasks with adapter and 35 mm 60 mm and 100 mm Petri dishes with adapter Maximum Plate Height 1 0 Hardware and Environmental Light Source Absorbance Fluorescence Xenon flash light source 20W maximum average FI monochromator based power not user changeable Fluorescence FI FP filter Xenon flash light source 5W maximum average based power not user changeable TRF filter based Xenon flash light source 5W maximum average power not user changeable 20 25 Dx 15 50 W x 17 5 H Dimensions 51 4 cm D x 39 4 cm W x 44 5 cm H Weiaht With all modules installed without power supply or Sid dispense module attached lt 80 lbs 36 3 kg Power up temperature 18 C to 30 C 64 4 F to
74. needs to be shipped again 6 Install the Power Supply Power Rating The instrument must be connected to a power receptacle that provides voltage and current within the specified rating for the system Use of an incompatible power receptacle may produce electrical shock and fire hazards Electrical Grounding Never use a plug adapter to connect primary power to the instrument Use of an adapter disconnects the utility ground creating a severe shock hazard Always connect the system power cord directly to an appropriate receptacle with a functional ground 1 Locate the power inlet on the back of the reader Examine the power supply s plug It has a small groove that lines up with a tab inside the power inlet 3 Insert the plug into the power inlet and plug the power supply s cord into an appropriate power receptacle Cytation 5 8 Installation Do not plug the power supply into a power receptacle until after the power supply is connected to the instrument 7 Install the Gas Controller if applicable The gas controller is an external module that enables the user to control CO and O concentrations inside the attached instrument s reading chamber If you purchased the module for operation with the Cytation 5 refer to the Gas Controller User Guide for installation instructions 8 Unpack and Install the Joystick if applicable If applicable 1 Open the shipping box lift out the joystick and place it on a level surf
75. nergy Low Run Time 00 00 45 Filter Sets Read Speed G a Kinetic Loop Interval 00 00 03 Reads 16 Step Label Sensitivity Read Buffer Read Well A6 C6 Filter Sets Filters EX 360 40 nm 620 40 nm 1 filter cube Optics Position Top 400 nm Auto Use first filter set gain from FIRST Read Step Normal Read Speed Delay after plate movement 100 msec Measurements per data point 50 Lamp Energy Low G Cytation 5 82 Instrument Testing Cytation 5_M_FI_T_SF prt and Cytation 5_M_FI_B_SF prt Default Setting Top Costar 96 black opaque Bottom Greiner SensoPlate Read Step 1 Detection Method Fluorescence Plate Type UU y y 1 ct MD Read Type Endpoint Run Time 00 00 45 Interval 00 00 3 Kinetic Loop Reads 16 Step Label Sensitivity Read Wavelength 1 EX 485 14 EM 528 14 nm Optics Position Top Bottom Auto Scale to High Wells D7 30000 Normal Delay after plate movement 100 msec Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Read Speed Detection Method Fluorescence Read Type Endpoint Run Time 00 01 35 Kinetic Loop Interval 00 00 6 Reads 16 Step Label Sensitivity Read Buffer Read Well C9 E9 Wavelength 1 EX 485 14 EM 528 14 nm Optics Position Top Bottom Auto Use first filter set gain from FIRST Read Step G G y mD BioTek Instrume
76. ntaminated instruments Gloved hands should be considered contaminated at all times keep gloved hands away from eyes mouth nose and ears BioTek Instruments Inc Clean Exposed Surfaces 45 Warning Mucous membranes are considered prime entry routes for infectious agents Wear eye protection and a surgical mask when there is a possibility of aerosol contamination Intact skin is generally considered an effective barrier against infectious organisms however small abrasions and cuts may not always be visible Wear protective gloves when handling contaminated instruments Caution The buildup of deposits left by the evaporation of spilled fluids within the read chamber can impact measurements Be sure to keep System Test records before and after maintenance so that changes can be noted Warning The instrument with all available modules weighs up to 80 Ibs 36 3 kg Use two people when lifting and carrying the instrument Clean Exposed Surfaces Exposed surfaces may be cleaned not decontaminated with a cloth moistened not soaked with water or water and a mild detergent You ll need e Deionized or distilled water e Clean lint free cotton cloths e Mild detergent optional e Canned air 1 Turn off and unplug the instrument Moisten a clean cotton cloth with water or with water and mild detergent Do not soak the cloth 3 Wipe the plate carrier and all exposed surfaces of the instrument 4 Instruments with
77. nts HyperClean hexamethyldisoloxan and ethanol Methyl ethyl ketone MEK available from Olympus ug le Isopropyl alcohol 70 30 with deionized water Dimethyl ketone acetone Methyl alcohol 70 30 with deionized water O Ethyl alcohol 70 30 with deionized water Cytation 5 50 Maintenance 1 From the Gen5 main screen go to System gt Instrument Configuration select Cytation 5 click View Modify gt Setup 2 In the Objective Configuration area in the Imaging Configuration tab click Access next to the desired objective to rotate the objective turret to the access position for that objective 3 Open the side door of the instrument Grasp one of the objectives unscrew it from the objective turret and remove it from the instrument 4 Inspect the lens using a magnifying glass if necessary to determine if there is dirt or dust present If so use a blower or a small paintbrush to remove any dirt and dust Any dirt or dust on the surface of the lens can cause extensive damage if dragged across the surface 5 Soak either an optical grade swab or a piece of lens cleaning tissue wrapped around tweezers in lens cleaning solvent or deionized water 6 Hold the swab or tissue wrapped tweezers still and rotate the objective s lens around it 7 Dry the lens immediately with a clean lens tissue 8 Replace the objective in the objective turret and screw it in to secure it 9 Repeat these steps to clean the second
78. nts Inc Fluorescence Liquid Tests 83 Default Setting Normal Delay after plate movement 100 msec Read Speed Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Detection Method Fluorescence Read Type Endpoint Read Wells A1 A3 A10 A12 H1 H3 H10 H12 Wavelength 1 EX 485 14 EM 528 14 nm Optics Position Top Bottom Auto Scale to High Wells A3 30000 Normal Delay after plate movement 100 msec Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Read Speed Detection Method Fluorescence Read Type Endpoint Step Label Linearity Read Read Wells C1 F5 Wavelength 1 EX 485 14 EM 528 14 nm Optics Position Top Bottom Auto Scale to High Wells C1 30000 UU D D 9 5 5 o 3 e et 4 Cytation 5 84 Instrument Testing Normal Delay after plate movement 100 msec Read Speed Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Luminescence Test BioTek provides two methods for verifying the performance of luminescence reads One method measures a Harta Luminometer Reference Microplate which is an LED based test plate Contact BioTek to purchase a plate or go to www hartainstruments com for more information The other method measures a LUX Biotechnology Ltd Glowell unit which is a small sealed cylinder with a gaseous tritium light source Before using the Cytation 5 F LumTest_Hart
79. o average 5 Horizontal offset from center of well Vertical offset from center of well Cytation 5 64 Instrument Testing Single image per well Enabled Cytation 5 AF_Reliability_4X prt Read Type Endpoint Plate Type 1222520_AF imaging qualification plate pe l Run Time 19 59 Interval 12 seconds Reads Kinetic Step 100 Objectives o o Rea melisa Focus method Auto focus with optional scan Minimum focus metric ratio 25 Scan distance 600 Auto Focus Options Scan increment 30 Minimum focus delta 8 of capture exposure for focus 75 Offset from bottom of well 50 Target exposure 75 Auto Exposure Options Skip 0 1 Integration threshold 100 Vibration Detection Options CV Threshold pa Images to average 5 Horizontal offset from center of well Vertical offset from center of well Single image per well Enabled Delay after plate movement O msec Cytation 5 AF_Reliability_10X prt Read Type Endpoint BioTek Instruments Inc Absorbance Liquid Test 65 Plate Type 1222520_AF imaging qualification plate gt l Run Time 19 59 Interval 12 seconds Reads Kinetic Step 100 Focus method Auto focus with optional scan Minimum focus metric ratio 50 Scan distance 600 Auto Focus Options Scan increment 30 Minimum focus delta 8 of capture exposure for focus 75 Offset from bottom of well 50 Target exposure 75 Auto Exposure Options Skip 0 1 Integration
80. o continue Dispenser lt select 1 or 2 depending on the protocol gt 5 Dispense Dispense to wells A9 H12 Tip prime before this dispense step 5 uL Dispense 5 uL at rate 225 uL sec Plate Suggested comment Weigh the plate 5 uL test RECORD the Out In weight PIPETTE 150 uL well of DI water into all 12 columns Place the plate back on the carrier Click OK to perform the Read steps Linear at 567 cpm 3 mm for 15 seconds Step label 80 ul Read_Disp 1 or _Disp 2 Wells A1 H4 Read Detection Method Absorbance Read Type Endpoint Read Speed Normal Two Wavelengths 405 and 750 nm Step label 20 and 5 ul Read_Disp 1 or _Disp 2 Wells A5 H12 Read Detection Method Absorbance Read Type Endpoint Read Speed Normal Two Wavelengths 630 and 750 nm Add Data Reduction Steps Each Read step is performed using two wavelengths Create two data reduction steps to calculate the Delta OD values 1 Select Protocol gt Data Reduction and select Custom 2 Within this dialog click Select Multiple Data Sets and then click DS2 Cytation 5 100 Instrument Testing e Set the Data In for DS1 to the 80 pl Read step at 405 nm e Set the Data In for DS2 to the 80 pl Read step at 750 nm 3 Click OK to return to the dialog 4 Inthe New Data Set Name field type an identifying name such as Delta OD 80 ul_Disp 1 5 Clear Use single formula for all wells 6 In the Current Formula field type DS1 DS2 and then assig
81. objective if necessary 10 Inthe Imaging Configuration tab on Reader Setup dialog which you opened in step 1 click Auto Calibration to calibrate the objectives When the calibration is finished the instrument is ready to use Tips e Do not allow the lens to air dry e Always use an unused portion of the lens tissue when wiping the lens e If smears are still present after performing these steps repeat the procedure Flush Purge the Fluid Path Applies only to Cytation 5 models with a dispenser At the end of each day that the dispense module is in use flush the fluid path using the Gen5 priming utility Leave the fluid to soak overnight or over a weekend and then purge the fluid before using the instrument again BioTek Instruments Inc Empty Clean the Tip Priming Trough 51 This flushing and purging routine is also recommended before disconnecting the outlet tubes from the reader and before decontamination to remove any assay residue prior to applying isopropyl alcohol or sodium hypochlorite To flush the fluid path 1 Fill two supply bottles with deionized or distilled water Insert the supply inlet tubes into the bottles 2 Place the priming plate on the carrier 3 From the Gen5 main screen select System gt Instrument Control gt Cytation 5 4 Click the Prime tab and select Dispenser 1 5 Set the Volume to 5000 pL Keep the default prime rate 6 Click Prime to start the process When the proc
82. or to use 3 Create an experiment based on the Cytation 5 AlphaTest_Crosstalk prt protocol Read the plate and then save the experiment Cytation 5 94 Instrument Testing 4 Open the Plate menu and export the data to the embedded Power Export spreadsheet The spreadsheet reports pass or fail for the test performed See Results Analysis next for descriptions of the calculations and troubleshooting tips 5 Print the spreadsheets and store them with your test records Results Analysis 1 Calculate the crosstalk for each of the four wells of Omnibead solution by dividing the background subtracted Mean value of the surrounding adjacent wells by the background subtracted Omnibead suspension well 2 Average the crosstalk of the four test wells to determine level of crosstalk Crosstalk Mean 3 Verify that the crosstalk is less than or equal to 0 1 4 Calculate the signal to noise ratio SNR by using the following equation SNR signal mean background mean SQRT signal STD background STD 5 Verify that SNR is greater than or equal to 10 Troubleshooting Alpha Tests If the test fails please try the following suggestions If the test s continue to fail print the results and contact BioTek s Technical Assistance Center e Are the solutions fresh e Have the solutions been stored properly between 2 C and 6 C e Has the kit been exposed to excessive light in excess of 100 lux e If the Crosstalk
83. ot already selected 3 Highlight the wavelength s to be included in this test Select only those wavelengths most appropriate for your use of the reader 4 Optional Enter any Comments 5 Click Start Test 6 Place the Test Plate in the microplate carrier so that well Al is in the right rear corner of the carrier 7 Click OK to run the test 8 When the test is completed the results report appears Scroll through the report every result should show PASS Imaging Tests These procedures test the level position of the carrier and the reliability of the Auto Focus feature Required Materials e Imaging qualification plate PN 1222520 e 10X or lower objective e DAPI377 447 cube PN 1225100 e Gen5 protocols described beginning on page 61 Cytation 5 lt x gt X_CarrierLevel prt Cytation 5 AF_Reliability_ lt x gt X prt BioTek Instruments Inc Imaging Tests 59 Carrier Level Test This test determines how level the carrier is in relation to the imaging system This test may be run with any of the following objectives Meiji 2 5X Zeiss 2 5X 4X or 10X 1 A Place the imaging qualification plate on the carrier with well A1 facing up in the right rear corner of the carrier If you have not already done so create the protocol described on page 61 Create a new experiment in Gens using the lt x gt X_CarrierLevel prt protocol with lt x gt representing the lowest power objective you have available
84. otek in Website www biotek in BioTek Singapore Phone 65 65922100 Email singapore biotek com Website www biotek com iv Preface BioTek South Korea BioTek Switzerland Phone 82 0 2 5624740 Phone 41 41 2504060 Email korea biotek com Email info biotek ch Website Website www biotek ch www biotekinstruments co kr BioTek Taiwan BioTek United Kingdom UK Phone 886 2 26277725 Email CustomerCare biotek com Email infotaiwanObiotek com Website www biotek uk com Website www biotek com Instructions for Use Requirements This document fulfills the basic needs of persons operating this device according to the requirements of the In Vitro Diagnostic Directive for Instructions for Use Some of the device s higher level functions and features as well as certain detailed maintenance and qualification routines are described in the Cytation 5 Operator s Manual Intended Use Statement The Cytation 5 is a hybrid multi mode microplate reader The performance characteristics of the data reduction software have not been established with any laboratory diagnostic assay The user must evaluate this instrument and PC based software in conjunction with their specific assay s This evaluation must include the confirmation that performance characteristics for the specific assay s are met e Ifthe instrument has an IVD label it may be used for clinical and non clinical purposes including research and development If ther
85. p priming trough in the rear pocket of the carrier 3 Place the priming plate on the carrier tip priming trough BioTek Instruments Inc 17 Run a System Test 23 4 Fill the two reagent bottles with distilled or deionized water Place the bottles in their holders and place the holders directly in front of the syringes Insert the inlet tubes into the bottles 5 From the Gen5 main screen select System gt Instrument Control gt Cytation 5 6 Click the Prime tab 7 With Dispenser set to 1 set the Volume to 5000 pL and click Prime The syringe should move down and up repeatedly drawing fluid from the bottle The fluid should pump through the tubing and dispense into the priming plate Examine the fittings no leaks should be detected If leaks are detected tighten all fittings and repeat the prime If leaks are still detected contact BioTek s Technical Assistance Center 8 When the prime finishes set Volume to 2000 pL and click Purge to clear the fluid lines 9 Set Dispenser to 2 and repeat steps 7 and 8 10 When finished remove and empty the priming plate 11 Return to the Gen5 main screen 17 Run a System Test Running a system test will confirm that the reader is set up and running properly or will provide an error code if a problem is detected 1 Turn on the incubator e From the Gen5 main screen select System gt Instrument Control gt Cytation 5 e Click the Pre Heating tab e Enter a Reque
86. pe tested by an independent accredited testing laboratory and found to meet the requirements of EN 61326 1 and EN 61326 2 6 for Immunity Verification of compliance was conducted to the limits and methods of the following EN 61000 4 2 Electrostatic Discharge EN 61000 4 3 Radiated EM Fields EN 61000 4 4 Electrical Fast Transient Burst EN 61000 4 5 Surge Immunity EN 61000 4 6 Conducted Disturbances from RFI EN 61000 4 8 Power Frequency Magnetic Field Immunity Test EN 61000 4 11 Voltage Dips Short Interruptions and Variations Directive 2014 35 EU Low Voltage Safety The system has been type tested by an independent testing laboratory and was found to meet the requirements of this Directive Verification of compliance was conducted to the limits and methods of the following EN 61010 1 Safety requirement for electrical equipment for measurement control and laboratory use Part 1 General requirements EN 61010 2 081 Particular requirements for automatic and semi automatic laboratory equipment for analysis and other purposes EN 61010 2 010 Particular requirements for laboratory equipment for the heating of materials BioTek Instruments Inc Electromagnetic Interference and Susceptibility ix EN 60825 1 Safety of laser products Part 1 Equipment classification and requirements Directive 2012 19 EU Waste Electrical and Electronic Equipment Disposal Notice Dispose of the instrument according to
87. r Module 11 A Cytation 5 must be connected via USB to the computer and turned on to appear in the Available Plug amp Play Readers list 5 To test that Gen5 can communicate with the instrument click Test Communications If the communication attempt is successful Gen5 displays a success message Return to Gen5 s main screen Communication Errors If the communication attempt is not successful try the following e Is the reader connected to the power supply and turned on e Is the communication cable firmly attached to both the reader and the computer e Did you select the correct Reader Type in Gen5 e Did you install the USB driver software If you remain unable to get Gen5 and the reader to communicate with each other contact BioTek s Technical Assistance Center 14 Install the Imager Module Several steps are required to install the imager module 1 Install the FireWire card and driver Set up Gen for imaging 3 Install the objectives LED cubes and imaging filter cubes and run Auto Calibration Tools e Screwdriver Desktop computer users typically need a screwdriver to install the FireWire card e 3 32 hex or Allen wrench 1 FireWire Video Card Computer and Camera Setup BioTek supplies the required FireWire card PCI Express Card EEE 1394b for either a desktop computer or a laptop Follow the applicable instructions for your workplace then install the FireWire software driver on your computer
88. rom bottom of well O CV Threshold 0 01 Images to average 1 Horizontal ofset from center af well O Vertical offset from centerofwells 0S Run Time 18 00 Kinetic Step Interval 2 seconds Reads 10 Objective Meiji 2 5X Zeiss 2 5X 4X 10X Read Wells A1 A12 H1 H12 Color Channel Bright Field LED 5 Exposure Integration time 50 Gain 0 Focus method Auto focus with optional scan Minimum focus metric ratio 3 Scan distance 600 Auto Focus Options Scan increment 30 Vibration Detection Options Minimum focus delta 8 of capture exposure for focus 75 Offset from bottom of well O BioTek Instruments Inc Imaging Tests 63 CV Threshold 0 01 Images to average 1 Vibration Detection Options Horizontal ofse rom center of well 0 Vertical offset rom center ofwel 0 AutoFocus Reliability Tests Cytation 5 AF_Reliability_Meiji 2 5X prt Cytation 5 AF_Reliability_Zeiss 2 5X prt Read Type Endpoint Plate Type 1222520 AF imaging qualification plate Run Time 19 59 Interval 12 seconds Reads Kinetic Step 100 Focus method Auto focus with optional scan Minimum focus metric ratio 3 Scan distance 600 Auto Focus Options Scan increment 30 Minimum focus delta 8 of capture exposure for focus 75 Offset from bottom of well 50 Target exposure 75 Auto Exposure Options Skip 0 1 Integration threshold 100 Vibration Detection Options CV Threshold donk Images t
89. rt the process 8 When finished carefully remove the priming plate from the carrier and empty it If the priming plate is empty the prime volume was too low Purge To save reagent Gen5 provides the option to purge fluid from the system back into the supply bottle BioTek Instruments Inc Gen5 Software 41 1 In Genb select System gt Instrument Control gt Cytation 5 and click the Prime tab Select the Dispenser number 1 or 2 associated with the supply bottle Enter the desired purge Volume in uL e g 2000 Select a prime Rate in uL second eS a Click Purge to start the process Cytation 5 42 Getting Started BioTek Instruments Inc Maintenance 44 Maintenance Preventive Maintenance A general preventive maintenance regimen for all Cytation 5 models includes periodically cleaning all exposed surfaces and inspecting cleaning the objectives emission and excitation filters and mirrors if used For models with the external dispense module additional tasks include flushing purging the fluid path and cleaning the tip prime trough priming plate supply bottles dispense tubing and injectors Daily Cleaning for the Dispense Module To ensure accurate performance and a long life for the dispense module and injectors flush and purge the fluid lines with deionized DI water every day or after completing an assay run whichever is more frequent Some reagents may crystallize or harden after u
90. ructions and descriptions of these features Perform an Image Read You can create an Image Read step to be used alone or in combination with the monochromator or filter based optics of the Cytation 5 1 Create a new protocol and select the Plate Type Create a read step and select Image as the Read Method 3 Optional Enter a step label or unique name for this step Data sets of the reading results will use the label in online views reports and export files 4 Select the objective Define whether to read a full plate or specific wells 6 Define up to four channels to read a Defining the color filter b Select whether exposure is automatic or defined Define which wells are used for auto exposure for each channel if applicable d Define focus and exposure options if needed 7 Define the horizontal and vertical offset from the center of the well 8 Select whether to create a single image for each well an image montage or an image Z stack 9 Define advanced options if needed BioTek Instruments Inc Gen5 Software 39 After you have defined and saved the protocol you can create an experiment and read the plate oe AS Create an experiment using an existing protocol Select the protocol you created with the imaging read step and click OK Select a plate in the menu tree and read it When prompted give the file an identifying name When the read is complete images and measurement values appear i
91. s BioTek Instruments Inc Contact Information Customer Service and Sales Contact Information iii 888 451 5171 toll free in the U S 802 655 4740 outside the U S Internet www biotek com Phone Fax 802 655 7941 Email Global Service and Support customercare biotek com BioTek instrument service and repair is available worldwide at one of BioTek s International Service Centers and in the field at your location For technical assistance contact the Technical Assistance Center TAC at BioTek US World Headquarters To arrange for service or repair of your instrument contact the office nearest you BioTek US World Headquarters BioTek China Beijing Phone 802 655 4740 Toll Free 888 451 5171 Service Toll Free 800 242 4685 Email CustomerCare biotek com Service Email TACObiotek com www biotek com BioTek China Shanghai Phone 86 021 50435800 Email infochina biotek com Website www biotekchina com cn BioTek Germany European Coordination Center Phone 49 0 71369680 Email info biotek de Website www biotek de BioTek Japan Phone 81 0 3 5812 8109 Email infojapan biotek com Website www biotek com ja Cytation 5 Phone 86 10 85865569 Email infochina biotek com Website www biotekchina com cn BioTek France Phone 33 3 89206329 Email info biotek fr Website www biotek fr BioTek India Phone 91 22 66870046 Fax 91 22 28759944 Email biotek bi
92. s and analyze the data These instructions briefly describe how to create a protocol in Gen5 See the Gen5 Help system for complete instructions 1 Open a new protocol Open the Procedure dialog If prompted to select a reader select the Cytation 5 and click OK 3 Select a Plate Type Gen5 stores measurements and other characteristics for individual plate types in a database It is essential that you select or define the plate type to match the assay plate Otherwise results may be invalid Cytation 5 36 Getting Started For imaging reads you must also define the Bottom Elevation parameter for the plate or slide If the relevant measurements are not defined Gen5 will not allow the objective to move closer than 1 mm from the bottom of the carrier thus disabling Cytation 5 s focus capabilities 4 Add steps to the procedure for shaking or heating the plate dispensing fluid reading the plate and more Click Validate to verify that the reader supports the defined steps and then click OK Optionally perform the next steps to analyze and report the results 5 Open the Plate Layout dialog and assign blanks samples controls and or standards to the plate 6 Open the Data Reduction dialog to add data reduction steps Categories include Transformation Well Analysis Curve Analysis Cutoff and Validation 7 Create a report or export template via the Report Export Builders 8 Select File gt Save and give the
93. s or filters suggest cleaning them here are some guidelines e Use of oil free dry air or nitrogen under moderate pressure is the best method for removing excessive debris from an optical surface If the contamination is not dislodged by the flow of gas please follow the cleaning instructions below BioTek Instruments Inc Inspect Clean Mirrors 47 e The purpose of the cleaning solvent is only to dissolve any adhesive contamination that is holding debris on the surface The towel needs to absorb both the excessive solvent and entrap the debris so that it can be removed from the surface Surface coatings on dichroics are typically less hard than the substrate It is reasonable to expect that any cleaning will degrade the surface at an atomic level Consideration should be given as to whether the contamination in question is more significant to the application than the damage that may result from cleaning the surface In many cases the AR coatings that are provided to give maximum light transmission amplify the appearance of contamination on the surface Materials e 7 64 hex key e Linen or cloth gloves e Anhydrous reagent grade ethanol e Kimwipes e Magnifying glass e 100 pure cotton balls for the polarizing filters Procedure 1 Open the access door on the front of the instrument and slide the filter cube straight out of its compartment 2 Set the filter cube on the work surface Using a 7 64 hex key remove the screw
94. s procedure follows the sequence below After each dispense step the plate is ejected to allow the operator to weight it and then tare the balance e Dispense 80 pL dye to columns 1 4 e Dispense 20 pL dye to columns 5 8 e Dispense 5 pL dye to columns 9 12 e Shake the plate for 15 seconds e Read columns 1 4 at 405 750 nm calculate the Delta OD e Read columns 5 12 at 630 750 nm calculate the Delta OD The detailed procedure is described on the next page To add a step to the procedure click the appropriate button on the left side of the Procedure dialog and define the required parameters The comments suggested for use with the Plate Out In steps are optional Gen5 Procedure Steps Details Dispenser lt select 1 or 2 depending on the protocol gt Dispense to wells A1 H4 Tip prime before this dispense step 20 uL Dispense 80 uL at rate 275 uL sec Suggested comment Weigh the plate 80 uL test RECORD the BioTek Instruments Inc Dispense Module Tests 99 Gen5 Procedure Steps Details weight TARE the balance Place the plate back on the carrier Click OK to continue Dispenser lt select 1 or 2 depending on the protocol gt Dispense to wells A5 H8 3 Dispense _ ae Tip prime before this dispense step 20 uL Dispense 20 uL at rate 250 uL sec Plate Suggested comment Weigh the plate 20 uL test RECORD the 4 Out In weight and TARE the balance Place the plate back on the carrier Click OK t
95. s the camera determine exposure settings and capture images LED Cubes and Imaging Filter Cubes The LED cubes and imaging filter cubes are located behind a door on the left side of the instrument and are user changeable Objectives The objectives are located next to the LED cubes and imaging filter cubes in an objective turret Gen5 supports the installation of up to six user changeable objectives Imaging Modes The Gen5 imaging module provides two modes of use manual and experiment e Manual mode allows you to view capture and analyze images outside of a protocol or experiment Images are displayed in real time You can also retrieve previously captured and saved images to analyze in manual mode e In experiment mode you can perform an image read step as an endpoint read or include it in a kinetic block in your procedures and experiments An imaging procedure can include additional steps as supported by the reader such as dispense shake incubate and different detection modes Change the Virtual Memory Settings You may find that you need more hard disk space than is allocated by default For example running a multiplate experiment imaging all 96 wells in a 4x4 montage in three colors will require an increase of virtual memory Without this change the message This procedure may require that you increase the size of the virtual memory in Windows may appear In this case please consult with your IT group to incr
96. se and clog the fluid passageways Take special care when using molecules that are active at very low concentrations e g enzymes inhibitors Remove any residual reagent in the dispense lines using a suitable cleaning solution review the reagent s package insert for specific recommendations Flushing the tubing at the end of each day letting the DI water soak overnight and then purging the lines at the beginning of each day ensures optimal performance of the dispense system BioTek recommends performing a visual inspection of the dispense accuracy before running an assay protocol that includes dispense steps Models with injectors Accumulated algae fungi or mold may require decontamination See the Cytation 5 Operators Manual for complete decontamination instructions Warnings and Precautions Read the following before performing any maintenance procedures Warning Internal Voltage Turn off and unplug the instrument for all maintenance and repair operations Important Do not immerse the instrument spray it with liquid or use a wet cloth on it Do not allow water or other cleaning solution to run into the interior of the instrument If this happens contact BioTek s Technical Assistance Center Important Do not apply lubricants to the microplate carrier or carrier track Lubricant attracts dust and other particles which may obstruct the carrier path and cause errors Warning Wear protective gloves when handling co
97. splays information about the camera Troubleshooting Communication with the Camera e Have you established communication with the instrument first e Did you install the FireWire software driver e Is the FireWire cable installed Is your laptop card plugged into an electrical outlet e Is the LED light on the back of the reader lit up e Red Power to the camera from the FireWire card e Half Red Half Green Ready state e Green Communicating activity Cytation 5 16 Installation 2 Set Up Gen5 for Imaging Configuration information must be set before any optical components are installed Use Gen to update the instrument s onboard settings The LED cubes and imaging filter cubes must be installed with the reader off Follow these installation steps in the order in which they are given di In Gen5 select System gt Instrument Configuration gt Cytation 5 gt View Modify gt Setup On the Imaging Configuration tab input the objective and LED cube and imaging filter cube configurations see 3a Setting the Objective LED Cube and Imaging Filter Cube Configuration on page 17 then click Send Values Click Access next to an objective position in the Objective Configuration area to lower the focusing system into the desired access position and to rotate the objective turret to the selected objective s installation position Install the objectives see 3b Install the Objectives on page 17 in their defined locations
98. sted temperature of at least 37 C and click On Wait until the incubator temperature reaches the set point before continuing 2 Return to Gen5 s main view and select System gt Diagnostics gt Run System Test If prompted to select a reader select Cytation 5 and click OK 3 When the test is completed a dialog requesting additional information appears Enter the information and click OK If a message appears that a pending system test is waiting from the initial power up self test view the pending system test and repeat steps 2 and 3 Cytation 5 24 Installation 4 The results report appears Scroll down toward the bottom the text should read SYSTEM TEST PASS e You may wish to print the report and store it with your records e The Gen software stores system test information in its database you can retrieve it at any time 5 Turn off the incubator e Select System gt Instrument Control gt Cytation 5 e Click the Pre Heating tab and click Off Repackaging and Shipping Instructions Refer to the Cytation 5 Operator s Manual for complete instructions for repackaging and shipping the instrument and accessories If the reader and or dispenser has been exposed to potentially hazardous material decontaminate it to minimize the risk to all who come in contact with the reader during shipping handling and servicing Decontamination prior to shipping is required by the U S Department of Transportation regulations Se
99. t must be lt 500 amol to pass To determine which PMT is installed check the label on the back of the reader 49984 low noise PMT 49721 red shifted PMT BioTek Instruments Inc Luminescence Test 87 Glowell Test Materials e Glowell PN GLO 466 formerly available from LUX BioTechology Ltd www luxbiotech com e Glowell Adapter Plate available from BioTek PN 7160006 e Filter cube PN 8040553 LUM Filter Block or a filter cube with a plug in EX position 1 or 2 and a hole in the corresponding EM position e Gen5 protocols described starting on page 88 Procedure 1 If you have not already done so insert the Glowell window side up into well D8 of the Adapter Plate 2 Create an experiment based on the Cytation 5 F Lum Test_Glowell prt or Cytation 5 M LumTest_Glowell prt protocol and read the plate 3 Calculate and evaluate the results as described on page 87 Plate Layout s e p Js b i Ja se or eur Ju sur eur sur Jour eur sur Joe aur eur eur BUE BUE BUF BUF BUF BUF BUF BUF BUF MN MD a BUE BUF BUE a a Glowell Results Analysis 1 Locate these items on the Glowell s Calibration Certificate Calibration Date Radiant Flux pW Measurement Uncertainty of the Radiant Flux 2 Calculate the number of days between the Calibration Date and the date the test was performed 3 Correct the Glowell s Radiant Flux value for deterioration over time Rad
100. tlet tube s thumbscrew counterclockwise to disconnect it from the injector Clean the Dispense Tubes and Injectors Some reagents can crystallize and clog the tubing and injectors Daily flushing and purging can help to prevent this but more rigorous cleaning may be necessary if reagent has dried in the tubing or injectors To clean the dispense tubes soak them in hot soapy water to soften and dissolve any hardened particles Flush each tube by holding it vertically under a stream of water To clean the injectors 1 Gently insert the stylus into each injector tip to clear any blockages The stylus is stored in a plastic cylinder affixed to the rear of the dispense module 2 Stream water through the pipe to be sure it is clean If the water does not stream out try soaking in hot soapy water and then reinserting the stylus Be careful not to damage the injector tips A damaged tip might not dispense accurately Cytation 5 54 Maintenance BioTek Instruments Inc Instrument Testing 56 Instrument Testing System Test Each time the Cytation 5 is turned on it automatically performs a series of tests on the reader s motors lamp s the PMT s and various subsystems If all tests pass the microplate carrier is ejected and the green LED on the carrier switch remains on If any test results do not meet the internally coded Failure Mode Effects Analysis FMEA criteria established by BioTek the reader beeps repeated
101. to be shipped again Thread the injector tip holder with outlet tubing connected to both ports through the hole in the top of the reader Open the reader s top door and holding the injector tip holder by the tab insert the injector tips into the appropriate holes inside the reader A magnet located between the injector tips helps to guide the tips into place and secures them in the reader 10 11 LZ Place the tubing feed through cover over the hole in the top of the reader and finger tighten the thumbscrews to secure it Remove the two syringes from their protective boxes They are identical and interchangeable Install both syringes e Hold the syringe vertically with the threaded end at the top e Screw the top of the syringe into the bottom of the syringe valve Finger tighten only e Carefully pull down the bottom of the syringe until it rests inside the hole in the bracket e Passa thumbscrew up through this hole and thread it into the bottom of the syringe Hold the syringe to prevent it from rotating while tightening the thumbscrew Finger tighten only Cytation 5 10 Installation 13 Locate the dispenser cable Plug one end into the port on the left side of the dispenser Plug the other end into the Dispenser Port on the rear of the reader 14 Locate the injector tip cleaning stylus packaged in a small cylinder Attach the cylinder to the back of the dispenser for storage 10 Connect the Host Co
102. ts Inc Gen5 Software 35 Define the Fluorescence Filter Cube For F models filter fluorescence the filter cube s characteristics must be entered into Gen5 and downloaded to the reader Perform these steps before using the reader for the first time and again if you change the filter cube s contents or switch to a different cube 1 Select System gt Instrument Configuration Highlight the Cytation 5 and click View Modify Click Setup and then click the Filter Cube tab Enter a name for the filter cube Select Fluorescence Polarization Cube if applicable oo oS TA Enter a filter set name for Filter Set 1 and define the excitation mirror and emission settings e Band Pass a standard interference filter with a defined central wavelength and bandwidth e Long Pass cutoff filters that transmit longer wavelengths and block shorter wavelengths e Short Pass cutoff filters that transmit shorter wavelengths and block longer wavelengths e Plug indicates the presence of a plug e Hole indicates an empty location 6 Repeat step 5 for Filter Set 2 Protocols and Experiments In Gen a protocol contains instructions for controlling the reader and optionally instructions for analyzing the data retrieved from the reader At a minimum a protocol must specify the procedure for the assay you wish to run After creating a protocol create an experiment that references the protocol You ll run the experiment to read plate
103. ttery check Daur leur eur eur E eur jour leur eur F leur jour leur eur aur eur Cytation 5 86 Instrument Testing Cytation 5 M LumTest_Harta prt NN battery battery e check check F eur ue aur Bur sur sur sur sur sur sur sur Sur mur leur eur Bur eur sur sur sur sur sur sur eur CC O O O O O O IO IS IS IS Harta Plate Results Analysis Through a manual correlation process it was found that the system requires approximately 35 photons per attomole of ATP thus a conversion factor of 0 02884 attomole photon was applied to determine ATP concentration from the NIST data in photons s 1 On the Harta plate s Calibration Certificate locate the NIST measurement for the A2 position and convert it to attomoles A2 NIST measurement 0 02884 2 Determine if the plate s battery is still functioning properly If A8 gt 0 2 A7 the battery is good Otherwise it requires replacement A replacement battery is included with each Harta plate A new Spare battery will be supplied when the plate is recertified 3 Calculate the signal to noise ratio A2 Mean of the buffer cells 3 Standard deviation of buffer cells 4 Calculate the detection limit A2 NIST measurement in attomoles signal to noise ratio If the reader is equipped with the low noise PMT the detection limit must be lt 75 amol to pass If the reader is equipped with the red shifted PMT the detection limi
104. two red polarization solutions these are not used Excitation filter 485 20 nm installed Emission filter 528 20 nm installed 510 nm dichroic mirror and polarizers installed Time Resolved Fluorescence TRF Test 15 mL conical bottom polypropylene sample tube Excitation filter 360 40 nm installed Emission filter 620 40 nm installed 400 nm dichroic mirror installed A new clean 96 well solid white microplate such as Corning Costar 3912 The recommended test solution FluoSpheres carboxylate modified microspheres 0 2 um europium luminescent 2 pL is available from Invitrogen Corporation PN F20881 or from BioTek PN 7160011 Cytation 5 70 Instrument Testing Test Solutions Corners Sensitivity Linearity Tests If using BioTek s sodium fluorescein powder PN 98155 be sure to hold the vial upright and open it carefully the material may be concentrated at the top If a centrifuge is available spin down the tube before opening When diluting the sodium fluorescein powder in buffer it takes time for the powder to completely dissolve Allow the solution to dissolve for five minutes with intermittent vortexing before preparing the titration dyes Wrap the vial containing the stock solution in foil to prevent exposure to light Discard unused solution after seven days Discard any open unused buffer solution after seven days 1 The Sodium Borate solution does not require further preparation proceed to step 2
105. ung mit dem Gebrauchsanweisung beachten Netz Consultar las instrucciones de uso Conectado Consultare le istruzioni per uso Chiuso O Off Supply In vitro diagnostic medical device Arr t alimentation Dispositif m dical de diagnostic in Aus Trennung vom Netz vitro Desconectado Medizinisches In Vitro Aperto sconnessione dalla Diagnostikum rete di alimentazione Dispositivo m dico de diagn stico in vitro Dispositivo medico diagnostico in vitro A Warning risk of electric pd Separate collection for electrical shock and electronic equipment Attention risque de choc Les quipements lectriques et lectrique lectroniques font l objet d une Gef hrliche elektrische collecte s lective schlag Getrennte Sammlung von Elektro Precauci n riesgo de und Elektronikger ten sacudida el ctrica Recogida selectiva de aparatos Attenzione rischio di scossa el ctricos y electr nicos elettrica Raccolta separata delle apparecchiature elettriche ed elettroniche BioTek Instruments Inc Installation 2 Installation Package Contents pre 5 Operator s Manual delivered on USB flash 1321000 Power cord set specific to installation environment Europe Schuko USA International United Kingdom Australia New Zealand USB cable 75108 2 Phillips screwdriver 01188 9 64 hex wrench 01623 Models with the imaging module FireWire desktop interface 01604 OR 1220535 Power supply only 01062 FireWire cable
106. uorescence System Cytation 5_M_FI_T_SF prt Cytation 5_M_FI_B SF prt optics Filter Set Setup Corners Sensitivity and Linearity tests using the Top Corners Sensitivity and Linearity tests using the Bottom optics Before using the filter based fluorescence test protocols create the applicable filter sets shown below in Gen5 Green is used for sodium fluorescein tests Blue for Filter Set 1 Filter Set Name Green Wavelength Bandwidth Excitation Band Pass 485 20 Mirror Dichroic Y Top 510 nm Ex Min Max Em Min Max 440 505 s15 640 Wavelength Bandwidth Emission Band Pass y 528 20 Filter Set 1 Filter SetName TRF Wavelength Bandwidth Excitation Bandpass 360 40 Mirror Dichroic al Top 400 nm Ex Min Max Em Min Max 320 320 40 800 Wavelength Bandwidth Emission Band Pass x 620 a Filter Set Name Excitation Mirror Filter Set 1 Blue Wavelength Bandwidth a o Dichroic v Top 400 nm Ex Min Max Em Min Max l 0 00 Wavelength Bandwidth Band Pass 460 40 Fluorescence Polarization Cube Filter Set Name Excitation Mirror Emission Corners Sensitivity Linearity Tests e Buffer Filter Set 1 FP Wavelength Bandwidth Pas a Top 510 nm Ex Min Max Em Min Max 515 640 Wavelength Bandwidth Band Pass v 520 Dichroic NIST tr
107. us click to capture it To save the captured images click Review Save 3 Click G to adjust the brightness and contrast adjustments or E to manually align overlaid images to compensate for pixel shift then click Save Channel Brigh tness a i Contrast o x shift Y shift _ Description OOO Set to neutral settings similar to the default settings 5 If selected changes to brightness and contrast will apply to all color channels of the image 6 Reset to default settings 8 ___ Gens displays the number of pixels the channel is shifted _ 9 Reset to default settings Cytation 5 38 Getting Started Adjustments made using the B amp C brightness and contrast and Channel Shift dialogs are for display purposes only the changes do not affect the data measurements from the images Analyze Captured and Saved Images 1 With your captured or saved images displayed in the left panel of the Capture dialog click Review Save 2 If necessary adjust the brightness and contrast of the image Click Analyze po In the Analyze Tool select which type of analysis you want to perform and define the parameters for the analysis then click Start The results are displayed in the right pane Imaging Module Experiment Mode Applies only to models with the imaging module The following sections briefly describe how to use the Gen5 imaging module in experiment mode See the Gen5 Help for more complete inst

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