Biotek Cytation 5 Instructions For Use Manual Download Page 85

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Fluorescence Liquid Tests |

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Aspirate 150 μL from wells C2

–F2, and dispense into wells C3–F3. Mix the wells

using the pipette. Do not discard the tips.

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Aspirate 150 μL from wells C3

–F3, and dispense into wells C4–F4. Mix the wells

using the pipette. Do not discard the tips.

•

Aspirate 150 μL from wells C4

–F4, and dispense into wells C5–F5. Mix the wells

using the pipette. Do not discard the tips.

•

Aspirate 150 μL from wells C5

–F5, and discard the tips.

Fluorescence Polarization (FP) Test

•

Pipette 200 µL of the (green) polarization buffer (BUF) into wells A6–H6.

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Pipette 200 µL of the green high polarization reference (HPR) into wells A7–B7.

•

Pipette 200 µL of the green low polarization reference (LPR) into wells A8–H8.

BUF

HPR

LPR

BUF

HPR

LPR

BUF

LPR

BUF

LPR

BUF

LPR

BUF

LPR

BUF

LPR

BUF

LPR

Cytation 5

Summary of Contents for Cytation 5

Page 1: ...Cytation 5 Cell Imaging Multi Mode Reader Instructions for Use BioTek Instruments Inc January 2015 2015 PN 1321022 Revision A ...

Page 2: ...struments 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 doe...

Page 3: ...na Beijing Phone 802 655 4740 Toll Free 888 451 5171 Service Toll Free 800 242 4685 Email CustomerCare biotek com Service Email TAC biotek com www biotek com Phone 86 10 85865569 Email infochina biotek com Website www biotekchina com cn BioTek China Shanghai BioTek France Phone 86 021 50435800 Email infochina biotek com Website www biotekchina com cn Phone 33 3 89206329 Email info biotek fr Websit...

Page 4: ...ment 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 m...

Page 5: ...apter 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 manufact...

Page 6: ...r 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...

Page 7: ...aution Warranty Failure to follow 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...

Page 8: ...mmunity 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 D...

Page 9: ...uipment 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 interferenc...

Page 10: ...010 1 Safety requirements for electrical equipment for measurement control and laboratory use Part 1 General requirements 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 EN 61010 Standards see CE Mark starting on page viii BioTek Instruments Inc ...

Page 11: ...h und Wechselstrom Corriente continua y corriente alterna Corrente continua e corrente alternata Laser radiation Do not stare into beam Rayonnement laser Ne pas regarder dans le faisceau Laserstrahlung nicht in den strahl blicken Radiación de laser No mire fijamente al rayo Radiazione di laser Non stare nel fascio Earth ground terminal Borne de terre Erde Betriebserde Borne de tierra Terra di funz...

Page 12: ...izinisches In Vitro Diagnostikum Dispositivo médico de diagnóstico in vitro Dispositivo medico diagnostico in vitro Warning risk of electric shock Attention risque de choc électrique Gefährliche elektrische schlag Precaución riesgo de sacudida eléctrica Attenzione rischio di scossa elettrica Separate collection for electrical and electronic equipment Les équipements électriques et électroniques fo...

Page 13: ...Installation ...

Page 14: ...Models with the imaging module FireWire desktop interface OR FireWire laptop card and power supply 01604 1220535 Power supply only 01062 FireWire cable 1220538 Microplate slide holder 1220548 Isolation table 1220521 Objective adapter collar wrench 1222187 Objective setup plate 1222531 3 32 hex wrench 48570 Models with an external dispense module packed separately with the following accessories Inj...

Page 15: ...le 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 th...

Page 16: ...of space between the instrument s rear panel and any other object This space ensures proper air flow in and out of the instrument The reader is sensitive to extreme environmental conditions Avoid the following 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 ...

Page 17: ... 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 2 Place the isolation table in the selected installation location 3 Place the instrument on the table as shown next Cytation 5 ...

Page 18: ...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 5 Remove the Shipping Hardware 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 BioTek Instruments Inc ...

Page 19: ...all 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...

Page 20: ...er 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 surface 2 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 p...

Page 21: ...reader needs to be shipped again 8 Thread the injector tip holder with outlet tubing connected to both ports through the hole in the top of the reader 9 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 r...

Page 22: ...lease 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 rea...

Page 23: ... 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 2 Set up Gen5 for imaging 3 Install the objectives LED cubes and imaging filter cubes and run Auto Calibration ...

Page 24: ...th 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 3 Insert the PCI Express card aligning it with its slot 4...

Page 25: ... cover or any existing PCI Express card from your laptop 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 fall off Cytation 5 ...

Page 26: ...n your laptop 6 Plug the power cord into the card then plug the cord into a power outlet 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 C Program Files BioTek Gen5 2 07 BioTek Instruments Inc ...

Page 27: ...able 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 Instrument Configuration select Cytation 5 and then click View Modify 2 Click Camera Information If communication is successful Gen5 displays information about the camera Troubleshooting Communicatio...

Page 28: ...otate the objective turret to the selected objective s installation position 3 Install the objectives see 3b Install the Objectives on page 17 in their defined locations 4 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 S...

Page 29: ...e position or positions you want to define or select None if you are removing an objective from the instrument Click Access 4 In the 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 Fo...

Page 30: ...ectives 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 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...

Page 31: ...14 Install the Imager Module 19 Cytation 5 ...

Page 32: ...button to stop the beeping 3 If the Reader Setup dialog is not already visible on your screen go to System Instrument Configuration select Cytation 5 then click View Modify 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 ...

Page 33: ...et the calibration values in Gen5 1 In Gen5 go to System Instrument Configuration select your instrument and click View Modify 2 Click Setup and then select the Dispenser 1 tab 3 On the keyboard press CTRL SHIFT M to enter maintenance mode for the Dispenser 1 window 4 Enter the syringe calibration values from the label on the rear of the dispenser box See Install the Dispenser 5 Click Send Volumes...

Page 34: ...t the Dispenser 1 If necessary press the carrier eject button to extend the microplate carrier 2 Place the tip priming trough in the rear pocket of the carrier 3 Place the priming plate on the carrier BioTek Instruments Inc ...

Page 35: ...e 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 From the Gen5 main screen select System Instrument Control ...

Page 36: ...efore shipment Spilled fluids can contaminate the optics and damage the instrument The instrument with all available modules weighs up to 80 lbs 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 As...

Page 37: ...Getting Started ...

Page 38: ... 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 ...

Page 39: ...hange the filters and mirrors yourself Note These cubes are not to be confused with the imaging LED cubes and filter cubes Injector System Applies to models with the dispense module The syringes may require replacement over time The tubing and injectors require cleaning at regular intervals Imaging System Applies to models with the imaging module The imaging system comprising a CCD camera objectiv...

Page 40: ...figuration Setup Select Band Pass Long Pass or Short Pass as appropriate for each filter type Band Pass a standard interference filter with a defined central wavelength and bandwidth Long Pass cutoff filters that transmit longer wavelengths and block shorter wavelengths Short Pass cutoff filters that transmit shorter wavelengths and block longer wavelengths Select PLUG to indicate the presence of ...

Page 41: ...tubes are short 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 op...

Page 42: ...s require a fluid reservoir to be present on the microplate carrier The priming plate is placed on the microplate carrier for a Prime operation to prime the dispense system with fluid 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 th...

Page 43: ...dure 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 ...

Page 44: ...nd 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 clickin...

Page 45: ... 12 109 6 9 1 548 26 3 18 141 3 7 1 425 27 4 24 169 0 5 9 355 28 5 30 195 4 5 1 307 29 6 36 222 8 4 5 269 30 Maximum Shaking Amplitude Based on Assay Volume and Plate Type 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 acc...

Page 46: ...6 mm No 1 6 mm No 48 well Microplate 0 0 5 mL 1 6 mm 1 6 mm 1 6 mm 1 6 mm 1 6 mm 0 5 1 mL 1 mm 1 6 mm 1 6 mm 1 6 mm 1 6 mm 1 1 3 mL No 1 6 mm 1 6 mm 1 6 mm No 96 well Microplate 0 0 25 mL 1 6 mm 1 6 mm 1 6 mm 1 6 mm 1 6 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 st...

Page 47: ...filters that transmit shorter wavelengths and block longer wavelengths Plug indicates the presence of a plug Hole indicates an empty location 6 Repeat step 5 for Filter Set 2 Protocols and Experiments In Gen5 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...

Page 48: ...ve and give the 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 1 Open a new experiment 2 Select the desired protocol and click OK 3 Select a plate in the menu tree and read it 4 When the read is complete measurement values appear in Gen5 Select the desired data set from the Da...

Page 49: ...rightness and contrast adjustments or to manually align overlaid images to compensate for pixel shift then click Save Description 1 Select a color channel 2 Use the sliders or buttons to adjust brightness and contrast 3 Have Gen5 automatically adjust brightness and contrast 4 Set to neutral settings similar to the default settings 5 If selected changes to brightness and contrast will apply to all ...

Page 50: ...tructions 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 2 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 w...

Page 51: ...ow the objective However the sample on your slide may be in a different location on the slide To find the sample a Select your lowest power objective b 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 ...

Page 52: ...ean 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 2 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 Gen5 select System Instrument Control Cytation 5 and click the Prime tab 4 Select t...

Page 53: ...ument Control Cytation 5 and click the Prime tab 2 Select the Dispenser number 1 or 2 associated with the supply bottle 3 Enter the desired purge Volume in µL e g 2000 4 Select a prime Rate in µL second 5 Click Purge to start the process Cytation 5 ...

Page 54: ...42 Getting Started BioTek Instruments Inc ...

Page 55: ...Maintenance ...

Page 56: ...d 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 Operat...

Page 57: ...can be noted Warning The instrument with all available modules weighs up to 80 lbs 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 Deionized or distilled water Clean lint free cotton cloths Mild detergent optional Canned ai...

Page 58: ...ly 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 ...

Page 59: ...face In many cases the AR coatings that are provided to give maximum light transmission amplify the appearance of contamination on the surface Materials 7 64 hex key Linen or cloth gloves Anhydrous reagent grade ethanol Kimwipes Magnifying glass 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...

Page 60: ...ble 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 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 b...

Page 61: ...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 Air puffe...

Page 62: ...leaning 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 objective if necessary 10 In the Imaging Configuration tab on Reader Setup dialog which you opened in st...

Page 63: ...sed 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 1 Place the inlet tubes in empty supply bottles or a beaker 2 Select System Instrument Control Cytation 5 3 Click the Prime tab and select Dispenser 1 4 Set the Volume to 2000 µL 5 Click Purge to start the process 6 When the purge is complete repea...

Page 64: ... 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 f...

Page 65: ...ectors 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 inje...

Page 66: ...54 Maintenance BioTek Instruments Inc ...

Page 67: ...Instrument Testing ...

Page 68: ...n 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 tu...

Page 69: ...wo Spectral Band Pass tables The Cytation 5 has a band pass wider than 5 nm for wavelengths greater than 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 certifica...

Page 70: ...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 A1 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 Imagi...

Page 71: ...reads in the Normal position in wells A1 A12 H1 and H12 2 Calculate the Mean focus height for each set of ten reads in the Turnaround position in wells A1 A12 H1 and H12 3 Compare the Mean values for each well in its Normal and Turnaround positions by performing these calculations a 1 A1 Normal Mean H12 Turnaround Mean b H12 Normal Mean A1 Turnaround Mean c 1 A12 Normal Mean H1 Turnaround Mean d H...

Page 72: ...a point compare the absolute value of the Residual with the objective s Limit µm 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 µm λ nm DOF µm Limit µm 10X 10 0 3 6 45 377 6 3 12 7 4X 4 0 13 6 45 377 34 7 69 4 Zeiss 2 5X 2 74 0 12 6 45 377 45 8 91 6 Me...

Page 73: ... 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 Detection Method Imaging Re...

Page 74: ...ffset from center of well 0 Single image per well Selected Delay after plate movement 300 msec After Read Step 1 Plate Out In Please rotate the plate 180 degrees Read Step 2 Process Mode Well Mode Kinetic Step Run Time 18 00 Interval 2 seconds Reads 10 Objective Meiji 2 5X Zeiss 2 5X 4X 10X Read Wells A1 A12 H1 H12 Color Channel Bright Field Exposure LED 5 Integration time 50 Gain 0 Auto Focus Opt...

Page 75: ...late Type 1222520_AF imaging qualification plate Kinetic Step Run Time 19 59 Interval 12 seconds Reads 100 Objective Meiji 2 5X Zeiss 2 5X Read Wells F7 Color Channel DAPI 377 447 Exposure Auto Auto Focus Options Focus method Auto focus with optional scan Minimum focus metric ratio 3 Scan distance 600 Scan increment 30 Minimum focus delta 8 of capture exposure for focus 75 Offset from bottom of we...

Page 76: ...ons Focus method Auto focus with optional scan Minimum focus metric ratio 25 Scan distance 600 Scan increment 30 Minimum focus delta 8 of capture exposure for focus 75 Offset from bottom of well 50 Auto Exposure Options Target exposure 75 Skip 0 1 Integration threshold 100 Vibration Detection Options CV Threshold 0 01 Images to average 5 Horizontal offset from center of well 0 Vertical offset from...

Page 77: ...ration Detection Options CV Threshold 0 01 Images to average 5 Horizontal offset from center of well 0 Vertical offset from center of well 0 Single image per well Enabled Delay after plate movement 0 msec 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...

Page 78: ...oTek s wetting agent 4 Fill to 1 liter with DI water cap and shake well The solution should measure approximately 2 000 OD when using 200 µL 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 2 Pipette 200 µL of the concentrated sol...

Page 79: ... that is compare A1 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 Deionized or distilled ...

Page 80: ...test protocols create the applicable filter sets shown below in Gen5 Green is used for sodium fluorescein tests Blue for MUB Corners Sensitivity Linearity Tests Buffer NIST traceable 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 indica...

Page 81: ...ecommended 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 two red polarization solutions these are not used Excitation filter 485 20 nm installed Emission filter...

Page 82: ...pare the solution Optional but recommended Using a 0 45 micron filter filter 200 mL of deionized or distilled water Follow the manufacturer s instructions on the PBS packaging to create 200 mL dissolving the necessary amount of PBS into the filtered water Stir the solution preferably using a stir table until the PBS is completely dissolved Check the pH it should be between 7 2 and 7 6 at 25 C 2 Pr...

Page 83: ... 15 mL conical bottom polypropylene sample tube This yields a 20 pM equivalent suspension 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 ...

Page 84: ... 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 Pipette 200 μL of the 3 3 nM SF solution into wells...

Page 85: ...se into wells C5 F5 Mix the wells using the pipette Do not discard the tips Aspirate 150 μL from wells C5 F5 and discard the tips Fluorescence Polarization FP Test Pipette 200 µL of the green polarization buffer BUF into wells A6 H6 Pipette 200 µL of the green high polarization reference HPR into wells A7 B7 Pipette 200 µL of the green low polarization reference LPR into wells A8 H8 1 2 3 4 5 6 7 ...

Page 86: ... 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 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 C...

Page 87: ...f the 500 pM wells 250 Mean of the 250 pM wells 125 Mean of the 125 pM wells 62 5 Mean of the 62 5 pM wells 3 Calculate the R Square value it must be 0 950 to pass Fluorescence Polarization FP Test 1 Using the raw data from the Parallel read Calculate the Mean Blank wells A6 H6 Calculate the Signal for each HPR well Subtract the Mean Blank from its measurement value Calculate the Signal for each L...

Page 88: ...dard Deviation of the PLPR must be less than Top with 510 nm dichroic mirror 5 mP 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...

Page 89: ...ells in the center of the plate may be slightly distorted due to the plate molding process and this can affect the standard deviation 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 soluti...

Page 90: ...ad Type Endpoint Kinetic Loop Run Time 00 00 45 Interval 00 00 03 Reads 16 Step Label Sensitivity Read Read Well D7 Filter Set 1 Green Filters EX 485 20 nm EM 528 20 nm Optics Position Top 510 nm Gain Auto Scale to High Wells D7 30000 Read Speed Normal Delay after plate movement 350 msec Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Read Height 7 00 mm Read Step 2 Detection...

Page 91: ... Endpoint Step Label Corners Read Read Wells A1 A3 A10 A12 H1 H3 H10 H12 Filter Set 1 Green Filters EX 485 20 nm EM 528 20 nm Optics Position Top 510 nm Gain Auto Scale to High Wells A3 30000 Read Speed Normal Delay after plate movement 350 msec Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Read Height 7 00 mm Read Step 4 Detection Method Fluorescence Read Type Endpoint Ste...

Page 92: ...ndpoint Plate Type Costar 96 black opaque 3915 Shake Step Linear for 15 seconds Delay Step 5 seconds after shake Synchronized Mode Plate Mode with Timing Control Read Wells A6 H8 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 Read Speed Normal Delay after plate movement 350 msec Measurements per data point 60 Lamp Energ...

Page 93: ...ment 100 msec Measurements per data point 50 Lamp Energy Low Read Height 7 00 mm Read Step 2 Detection Method Time resolved fluorescence Read Type Endpoint Kinetic Loop Run Time 00 00 45 Interval 00 00 03 Reads 16 Step Label Sensitivity Read Buffer Read Well A6 C6 Filter Sets 1 filter cube Filters EX 360 40 nm 620 40 nm Optics Position Top 400 nm Gain Auto Use first filter set gain from FIRST Read...

Page 94: ...EM 528 14 nm Optics Position Top Bottom Gain Auto Scale to High Wells D7 30000 Read Speed Normal Delay after plate movement 100 msec Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Read Height for top optics 7 00 mm Read Step 2 Detection Method Fluorescence Read Type Endpoint Kinetic Loop Run Time 00 01 35 Interval 00 00 6 Reads 16 Step Label Sensitivity Read Buffer Read Well...

Page 95: ...ls A1 A3 A10 A12 H1 H3 H10 H12 Wavelength 1 EX 485 14 EM 528 14 nm Optics Position Top Bottom Gain Auto Scale to High Wells A3 30000 Read Speed Normal Delay after plate movement 100 msec Measurements per data point 50 Lamp Energy Low Dynamic Range Standard Read Height for top optics 7 00 mm Read Step 4 Detection Method Fluorescence Read Type Endpoint Step Label Linearity Read Read Wells C1 F5 Wave...

Page 96: ...o 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_Harta prt or Cytation 5 F LumTest_Glowell prt protocols described in this section create the filter set shown below Harta Plate Test Materials Harta Luminometer Reference Microplate ...

Page 97: ...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 Cyta...

Page 98: ...o attomoles A2 NIST measurement 0 02884 2 Determine if the plate s battery is still functioning properly If A8 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 ...

Page 99: ...M LumTest_Glowell prt protocol and read the plate 3 Calculate and evaluate the results as described on page 87 Plate Layout 1 2 3 4 5 6 7 8 9 10 11 12 A BUF BUF BUF B BUF BUF BUF C BUF BUF BUF D Glowell BUF BUF BUF E BUF BUF BUF F BUF BUF BUF G BUF BUF BUF H BUF BUF BUF Glowell Results Analysis 1 Locate these items on the Glowell s Calibration Certificate Calibration Date Radiant Flux pW Measureme...

Page 100: ...t 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 75 amol to pass If the reader is equipped with the red shifted PMT the detection limit must be 500 amol to pass Gen5 Protocol Reading Parameters Cytation 5 F LumTest_Harta prt Parameter Defau...

Page 101: ...ndard Read Height 7 00 mm Read Step 3 Detection Method Luminescence Read Type Endpoint Step Label Battery Check Read Wells A7 A8 Filter Sets 1 Open Excitation Plug Emission Hole Gain 60 Integration Time 0 01 00 MM SS ss Delay after plate movement 350 msec Dynamic Range Standard Read Height 10 00 mm Cytation 5 M LumTest_Harta prt Parameter Default Setting Plate Type 8030015 Harta w o 8032028 adapte...

Page 102: ...escence Read Type Endpoint Step Label Background Read Wells F1 G12 Wavelength 1 Gain 150 Integration Time 0 10 00 MM SS ss Delay after plate movement 350 msec Dynamic Range Standard Read Height 1 00 mm Read Step 3 Detection Method Luminescence Read Type Endpoint Step Label Battery Check Read Wells A7 A8 Wavelength 1 Gain 80 Integration Time 0 01 00 MM SS ss Delay after plate movement 350 msec Dyna...

Page 103: ...e Gain 150 Integration Time 0 10 00 MM SS ss Delay after plate movement 350 msec Dynamic Range Standard Read Height 7 00 mm Cytation 5 M LumTest_Glowell prt Parameter Default Setting Plate Type Costar 96 well black opaque 3915 Delay Step 3 minutes Detection Method Luminescence Read Type Endpoint Read Wells A8 H11 Wavelenths 1 Gain 150 Integration Time 0 10 00 MM SS ss Delay after plate movement 35...

Page 104: ...m 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 µL well volumes Required Materials Recommended test solution Alphascreen Omnibead Assay kit available...

Page 105: ...C Pipette Map 1 2 3 4 5 6 7 8 9 10 11 12 A BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF B BUF 20 µg mL OMB BUF BUF 20 µg mL OMB BUF BUF 20 µg mL OMB BUF BUF 20 µg mL OMB BUF C BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF D E F G BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF H BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF BUF Procedure Crosstalk Test 1 Pipette 100 µL of PBS in wells A1 A12 B1...

Page 106: ...nal 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 Are the solutions fresh Have the solutions been stored properly between 2 C and 6 C Has the kit been exposed to excessive light in excess of 100 lu...

Page 107: ...r 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 Microplate shaker if the absorbance reader does not support shaking Precision balance with capacity of 100 g minimum and readability of 0 001 g 50 200 µL hand pipette and disposable tips Deionized water Supply bottles 250 mL beaker New 96 well clear flat ...

Page 108: ... No 5 0 092 grams Tween 20 1 0 mL Sodium Azide N3Na 0 100 gram Deionized water Make to 1 liter Procedure The Cytation 5 Operator s Manual contains the procedure for models without absorbance capabilities 1 If you 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 µL of deionized or disti...

Page 109: ...well to columns 9 12 f Remove the plate and weigh it Record the weight g Manually pipette 150 µL of deionized or distilled water into all 12 columns on top of the green test dye solution h Place the plate on the carrier for a 15 second shake the 80 µL read at 405 750 nm and the 20 and 5 µL read at 630 750 nm 10 When processing is complete save the file with an identifying file name 11 Remove the p...

Page 110: ... weight it and then tare the balance Dispense 80 μL dye to columns 1 4 Dispense 20 μL dye to columns 5 8 Dispense 5 μL dye to columns 9 12 Shake the plate for 15 seconds Read columns 1 4 at 405 750 nm calculate the Delta OD 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 l...

Page 111: ...late Out In Suggested comment Weigh the plate 5 uL test RECORD the 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 7 Shake Linear at 567 cpm 3 mm for 15 seconds 8 Read Step label 80 ul Read_Disp 1 or _Disp 2 Wells A1 H4 Detection Method Absorbance Read Type Endpoint Read Speed Normal Two Wavelengths 405 and 750 nm 9 ...

Page 112: ...ngle formula for all wells 6 In the Current Formula field type DS1 DS2 and then assign 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 DS1 set to the 20 and 5 μL Read step at 630 nm DS2 set to the 20 and 5 μL Read step at 750 nm New Data Set Name resembling Delta OD 20 and ...

Page 113: ...Specifications ...

Page 114: ...ser changeable Dimensions 20 25 D x 15 50 W x 17 5 H 51 4 cm D x 39 4 cm W x 44 5 cm H Weight With all modules installed without power supply or dispense module attached 80 lbs 36 3 kg Environment Power up temperature 18ºC to 30ºC 64 4ºF to 86ºF Note Performance measurements including detection limits were verified up to 25ºC 77ºF Humidity 10 to 85 relative humidity non condensing Power Supply 24 ...

Page 115: ... 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 ...

Page 116: ...gent dispense module Plate Type Both injectors dispense to standard height 6 12 24 48 96 and 384 well microplates Detection Method Absorbance Fluorescence FI FP TRF Luminescence Imaging well mode only Volume Range 5 1000 µL with a 5 20 µL tip prime Reagent Dead Volume 1100 µL with dead volume recovery function back flush Injection Speeds 225 250 275 300 µL second Accuracy 1 µL or 2 0 whichever is ...

Page 117: ...Out Speed 35 seconds 450 nm sweep mode 96 well microplate Accuracy Linearity Repeatability Specifications apply from 250 999 nm 200 µL 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 OD 2 0 010 OD del...

Page 118: ...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 wel...

Page 119: ...ith low noise PMT 300 700 nm for emission scans with low noise PMT 250 900 nm with red shifted PMT 300 900 nm for emission scans with red shifted PMT Selectable increment 1 nm Bandpass Variable from 9 nm to 50 nm in 1 nm increments both excitation and emission Minimum kinetic interval 20 seconds sweep mode 96 well microplate Plate In Plate Out Speed 35 seconds sweep mode 96 well microplate Sensiti...

Page 120: ... 528 20 510 nm mirror DL 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 250 fM 100 fM typical Excitation 360 40 nm Emission 620 40 nm 400 nm mirror Integration time 20 to 2000 µs Delay 0 to 2000 µs Granularity 1 µs step Fluorescence Polarization 5 mP standard deviation at 1 nM Sodium Fluoresce...

Page 121: ... 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 Integration Time 10 seconds Gain 150 Blank Wells 16 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 pl...

Page 122: ...110 Specifications BioTek Instruments Inc ...

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