Biotek Cytation 5 Instructions For Use Manual
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Biotek Cytation 5 Instructions For Use Manual

Cell imaging multi-mode reader
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Cytation™ 5
Cell Imaging Multi-Mode Reader
Instructions for Use
BioTek
Instruments, Inc.
®
January 2015
© 2015
PN 1321022
Revision A

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  • Page 1 Cytation™ 5 Cell Imaging Multi-Mode Reader Instructions for Use BioTek Instruments, Inc. ® January 2015 © 2015 PN 1321022 Revision A...
  • Page 2 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 dealers.
  • Page 3 Email: customercare@biotek.com Global Service and Support 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.
  • Page 4 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.
  • Page 5 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...
  • Page 6 Precautions The following precautions are provided to help avoid damage to the instrument: Caution: Service. The instrument should be serviced by BioTek- authorized service personnel. Only qualified technical personnel should perform service procedures on internal components.
  • Page 7 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. Cytation 5...
  • Page 8 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.
  • Page 9 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...
  • Page 10 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 Protective conductor Caution (refer to accompanying terminal documents) Borne de terre de protection Attention (voir documents d'accompanement) Schultzleiteranschluss Achtung siehe Begleitpapiere Borne de tierra de protección Atención (vease los documentos incluidos) Terra di protezione Attenzione, consultare la doc annessa Cytation 5...
  • Page 12 électroniques font l'objet d'une collecte sélective Gefährliche elektrische schlag Getrennte Sammlung von Elektro- und Elektronikgeräten Precaución, riesgo de sacudida eléctrica Recogida selectiva de aparatos eléctricos y electrónicos Attenzione, rischio di scossa elettrica Raccolta separata delle apparecchiature elettriche ed elettroniche BioTek Instruments, Inc.
  • Page 13 Installation...

  • Page 14: Package Contents

    | Installation Package Contents Item Part # Cytation 5 Operator's Manual (delivered on USB flash 1321000 drive) Power cord set (specific to installation environment): Europe (Schuko) 75010 75011 USA/International United Kingdom 75012 Australia/New Zealand 75013 USB cable 75108 #2 Phillips screwdriver 01188 9/64"...
  • Page 15 1210009 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.
  • Page 16 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.
  • Page 17 4: Select an Appropriate Location | 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.
  • Page 18 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 2. 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...
  • Page 20 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...
  • Page 21 • Carefully pull down the bottom of the syringe until it rests inside the hole in the bracket. • Pass a 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...
  • Page 22 10: Connect the Host Computer 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.
  • Page 23 14: Install the Imager Module | A Cytation 5 must be connected via USB to the computer and turned on to appear in the Available Plug & Play Readers list. 5. To test that Gen5 can communicate with the instrument, click Test .
  • Page 24 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.
  • Page 25 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 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 Administrator a message appears: "SUCCESS: Installed package <path to package>". 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.
  • Page 28 Follow these installation steps in the order in which they are given. 1. In Gen5, select System > Instrument Configuration > Cytation 5 > View/Modify > Setup 2. On the Imaging Configuration tab, input the objective and LED cube and imaging filter cube configurations (see 3a.
  • Page 29 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 Gen5: 1.
  • Page 30 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.
  • Page 31 14: Install the Imager Module | Cytation 5...
  • Page 32 After the calibration procedure is finished, 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 > Instrument Configuration > Cytation 5 > View/Modify > Setup BioTek Instruments, Inc.
  • Page 33 4. 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 , select your System >...
  • Page 34 16: Test 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 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 and click to clear 2000 µL...
  • Page 36 • 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.
  • Page 37 Getting Started...
  • Page 38 | Getting Started Overview The Cytation 5 is a hybrid multi-mode microplate reader. 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;...
  • Page 39 Internal Components Component Description The filter cube can contain excitation and emission filters, mirrors, and polarizing filters. Preconfigured cubes are available from BioTek, or you can change the filters and mirrors yourself. Filter Cube Note: These cubes are not to be confused with the imaging LED cubes and filter cubes.
  • Page 40 | 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.
  • Page 41 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.
  • Page 42 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.
  • Page 43 , if it is selected. Automatically manage paging file size for all drives 6. Select , enter the following minimum and maximum values, and Custom Size then click • Initial size: 10 GB Maximum size: 20 GB • Cytation 5...
  • Page 44 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. BioTek Instruments, Inc.
  • Page 45 Sample Orbital Orbital Double Orbital Double Orbital Linear Volume Slow Fast Slow Fast 6-well Microplate 1–6 0–3 mL 1–6 mm 1–6 mm 1–6 mm 1–6 mm 1–6 3–4 mL 1–6 mm 1–6 mm 1–6 mm 1 mm Cytation 5...
  • Page 46 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 stacker (if equipped); perform data reduction and analysis on the measurement values;...
  • Page 47 1. Select . Highlight the System > Instrument Configuration Cytation 5 and click View/Modify 2. Click , and then click the tab.
  • Page 48 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.
  • Page 49 If selected, changes to brightness and contrast will apply to all color channels of the image Reset to default settings Select a color channel Gen5 displays the number of pixels the channel is shifted Reset to default settings Adjust the shift using the arrows Cytation 5...
  • Page 50 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 as the Read Method.
  • Page 51 Auto Exposure, then click to enter Auto manual mode. 3. Follow steps 2 and 3 in the manual mode procedure (above) to find your samples, then click . Gen5 imports your exposure settings Save settings Cytation 5...
  • Page 52 Insert the appropriate inlet tube into the bottle. 3. In Gen5, select and click System > Instrument Control > Cytation 5 tab. Prime 4. Select the Dispenser number (1 or 2) associated with the supply bottle.
  • Page 53 Gen5 Software | 1. In Gen5, select and click System > Instrument Control > Cytation 5 tab. Prime 2. Select the Dispenser number (1 or 2) associated with the supply bottle. 3. Enter the desired purge Volume in µL (e.g., 2000).
  • Page 54 | Getting Started BioTek Instruments, Inc.
  • Page 55 Maintenance...
  • Page 56 | 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.
  • Page 57 3. Wipe the plate carrier and all exposed surfaces of the instrument. 4. Instruments with 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...
  • Page 58 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.
  • Page 59 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...
  • Page 60 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 as a result.
  • Page 61 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.
  • Page 62 • 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.
  • Page 63 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.
  • Page 64 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 •...
  • Page 65 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...
  • Page 66 | Maintenance BioTek Instruments, Inc.
  • Page 67 Instrument Testing...
  • Page 68 | 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.
  • Page 69 • 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 than 285 nm and less than 4 nm for 230–285 nm.
  • Page 70 Focus feature. Required Materials • Imaging qualification plate, PN 1222520 • 10X or lower objective • DAPI 377/447 cube (PN 1225100) • Gen5 protocols described beginning on page 61 Cytation 5 <x>X_CarrierLevel.prt • Cytation 5 AF_Reliability_<x>X.prt • BioTek Instruments, Inc.
  • Page 71 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...
  • Page 72 FM Ratio [0:DAPI 377,447] Refer to the chart below for passing criteria for the value displayed in well Objective FM Ratio must be Meiji 2.5X > 1.4 Zeiss 2.5X > 1.4 > 25 > 50 BioTek Instruments, Inc.
  • Page 73 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...
  • Page 74 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: 0 BioTek Instruments, Inc.
  • Page 75 Horizontal offset from center of well: Vertical offset from center of well: Single image per well: Selected Delay after plate movement: 300 msec AutoFocus Reliability Tests Cytation 5 AF_Reliability_Meiji 2.5X.prt, Cytation 5 AF_Reliability_Zeiss 2.5X.prt Parameter Default Setting Detection Method: Imaging Read Type:...
  • Page 76 | Instrument Testing Parameter Default Setting Single image per well Enabled Delay after plate movement: 0 msec Cytation 5 AF_Reliability_4X.prt Parameter Default Setting Detection Method: Imaging Read Type: Endpoint Plate Type: 1222520_AF (imaging qualification plate) Run Time: 19:59, Interval: 12 seconds, Reads:...
  • Page 77 • 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 • BioTek QC Check Solution No. 1 (PN 7120779, 25 mL; PN 7120782, 125 mL) • Deionized water Cytation 5...
  • Page 78 • 5-mL Class A volumetric pipette • 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 µL in a flat-bottom microwell.
  • Page 79 • (Optional) Black polyethylene bag(s) to temporarily store plate(s) • 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...
  • Page 80 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 • Sodium Fluorescein Powder (1-mg vial, BioTek PN 98155) BioTek Instruments, Inc.
  • Page 81 The FP Test can be performed in conjunction with the top Corners/Sensitivity/Linearity Tests, in the same microplate. • 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 ...
  • Page 82 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.
  • Page 83 Fluorescence Liquid Tests | 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.
  • Page 84 • Pipette 150 μL of the 1 nM SF solution into wells C1–F1. • Pipette 150 μL of the 1 nM SF solution into wells C2–F2. Mix the wells using the pipette. Do not discard the tips. BioTek Instruments, Inc.
  • Page 85 • 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. Cytation 5...
  • Page 86 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.
  • Page 87 • 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 LPR well: Subtract the Mean Blank from its measurement value. Cytation 5...
  • Page 88 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’s Technical Assistance Center. BioTek Instruments, Inc.
  • Page 89 • Does the Plate Type setting in the Gen5 protocol match the plate you used? • 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.
  • Page 90 Dynamic Range: Standard Read Height: 7.00 mm Read Step 2 Detection Method: Fluorescence Read Type: Endpoint 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.
  • Page 91 Read Step 4 Detection Method: Fluorescence Read Type: Endpoint Step Label: "Linearity Read" Read Wells: C1..F5 Filter Set: 1 (Green) Filters: EX 485/20 nm, EM 528/20 nm Optics Position: Top 510 nm Gain: Auto, Scale to High Wells, C1, 30000 Cytation 5...
  • Page 92 Delay after plate movement: 350 msec Read Speed: Measurements per data point: 60 Lamp Energy: Low Read Height: 7.0 mm Cytation 5_TRF.prt Parameter Default Setting Plate Type: Costar 96-well white opaque Delay Step: 3 minutes Read Step 1 Detection Method: Time-resolved fluorescence BioTek Instruments, Inc.
  • Page 93 EX 360/40 nm, 620/40 nm Optics Position: Top 400 nm Gain: Auto, Use first filter set gain from FIRST Read Step Normal Delay after plate movement: 100 msec Read Speed: Measurements per data point: 50 Lamp Energy: Low Read Height: 7.00 mm Cytation 5...
  • Page 94 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 Gain Auto, Use first filter set gain from FIRST Read Step BioTek Instruments, Inc.
  • Page 95 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 Wavelength: 1, EX 485/14, EM 528/14 nm Optics Position: Top/Bottom Gain Auto, Scale to High Wells, C1, 30000 Cytation 5...
  • Page 96 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...
  • Page 97 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 Cytation 5 M-LumTest_Harta.prt protocol and read the plate. 5. Calculate and evaluate the results as described on page 86.
  • Page 98 | Instrument Testing Cytation 5 M-LumTest_Harta.prt battery battery meas check check G BUF 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.
  • Page 99 Materials • Glowell, PN GLO-466, formerly available from LUX BioTechology, Ltd. (www.luxbiotech.com) • Glowell Adapter Plate, available from BioTek, PN 7160006 • Filter cube PN 8040553, LUM Filter Block, a filter cube with a plug in EX position 1 or 2 and a hole in the corresponding EM position.
  • Page 100  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 Default Setting Plate Type: 8030015 Harta - w/o 8032028 adapter Delay Step:...
  • Page 101 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 adapter Delay Step: 3 minutes Read Step 1 Detection Method:...
  • Page 102 1.00 mm Read Step 3 Detection Method: Luminescence Read Type: Endpoint Step Label: "Battery Check" Read Wells: A7–A8 Wavelength Gain: Integration Time: 0:01.00 MM:SS.ss Delay after plate movement: 350 msec Dynamic Range: Standard Read Height: 1.00 mm BioTek Instruments, Inc.
  • Page 103 Luminescence Test | Cytation 5 F-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 Filter Sets: 1 (Open) Excitation: Plug Emission: Hole Gain: Integration Time: 0:10.00 MM:SS.ss...
  • Page 104 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 Efficiency Alpha Jig (PN 7162512 Rev F or higher).
  • Page 105 B11 (see pipette map, “20 µg/mL OMB” wells). Allow the plate to sit at room temperature for approximately 15 minutes prior 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...
  • Page 106 • Have the solutions been stored properly (between +2°C and +6°C)? • Has the kit been exposed to excessive light (in excess of 100 lux)? • If the Crosstalk test continues to fail, the laser may not be firing. Contact BioTek TAC.
  • Page 107 ± 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.
  • Page 108 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:...
  • Page 109 10. When processing is complete, save the file with an identifying file name. 11. Remove the plate from the carrier and set it aside. 12. Repeat steps 5–11 using Cytation 5 Disp 2 Test.prt. 13. See the instructions for analyzing the results below.
  • Page 110 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’s procedure follows the sequence below.
  • Page 111 Each Read step is performed using two wavelengths. Create two data reduction steps to calculate the Delta OD values. 1. Select and select Protocol > Data Reduction Custom 2. Within this dialog, click and then click Select Multiple Data Sets Cytation 5...
  • Page 112 20 and 5 μL Read step at 630 nm • set to the 20 and 5 μL Read step at 750 nm • • New Data Set Name resembling ‘Delta OD 20 and 5 ul_Disp <#>’ Formula DS1-DS2 applied to wells A5 to H12 • BioTek Instruments, Inc.
  • Page 113 Specifications...

  • Page 114: General Specifications

    | 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).
  • Page 115 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 ± 1 µL or 2.0%, whichever is greater ≤ 2.0% for volumes of 50–200 µL ≤ 4.0% for volumes of 25–49 µL Precision ≤ 7.0% for volumes of 10–24 µL ≤ 10.0% for volumes of 5–9 µL BioTek Instruments, Inc.
  • Page 117 384-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 Cytation 5...
  • Page 118 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 < 5 ng/µL BioTek Instruments, Inc.
  • Page 119 Fluorescence Specifications (Mono-Based) | 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, which 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.
  • Page 120 | 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 ≤ 35 seconds for filter set, sweep mode, 96-well microplate Fluorescence Intensity DL ≤ 10 pM (3 pM typical) solution of Sodium Fluorescein in PBS Excitation 485/20, Emission 528/20, 510 nm mirror DL ≤...
  • Page 121 Luminescence Specifications | 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 µL/well, at room temperature. Production testing is performed using a Harta plate. Luminescence ≤...
  • Page 122 | Specifications BioTek Instruments, Inc.

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