Agilent Technologies NovoCyte Quanteon Operator's Manual
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Agilent Technologies NovoCyte Quanteon Operator's Manual

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NovoCyte Quanteon Flow Cytometer
Operator's Guide
For Research Use Only. Not for use in diagnostic procedures.

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Summary of Contents for Agilent Technologies NovoCyte Quanteon

  • Page 1 NovoCyte Quanteon Flow Cytometer Operator's Guide For Research Use Only. Not for use in diagnostic procedures.
  • Page 2 Agilent disclaims all © Agilent Technologies, Inc. 2021 not correctly performed or adhered to, warranties, either express or implied, No part of this manual may be...

  • Page 3: In This Guide

    In This Guide In This Guide This guide covers following module: NovoCyte Quanteon Flow Cytometer and associated standard accessories. Prologue This chapter describes the purpose and scope of this guide, symbols used in this guide, glossary, safety and limitations, Agilent regulatory compliance statement and technical support information.
  • Page 4 Version History Appendix A: NovoCyte Quanteon Laser and Detection Channel Configuration This chapter provides additional information on NovoCyte Quanteon laser and detection channel configuration. Appendix B: NovoCyte Quanteon Technical Specifications This chapter provides technical specifications of NovoCyte Quanteon flow cytometer and associated standard accessories.

  • Page 5: Table Of Contents

    Electromagnetic Compatibility EMC Declaration for South Korea Detachable Power Cord Declaration for Japan Sound Emission Certification for Federal Republic of Germany Waste Electrical and Electronic Equipment (WEEE) Directive Regulatory Compliance Identification Number Technical Support Introduction NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 6 NovoCyte Fluidics Station II NovoCyte Fluidics Cart (Optional) NovoSampler Q (Optional) Installation Installation Warnings Space Requirements Environmental Requirements Instruction for Lifting and Carrying Install NovoCyte Quanteon Connect NovoCyte Workstation Connect NovoCyte Fluidics Station II NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 7 Modify Instrument Configuration Instrument QC Test Instrument Shutdown Running Samples Overview Create Experiment Define Sample Acquisition Parameters Set Sample Acquisition Conditions Stop Condition Flow Rate Threshold Run Sample Perform Fluorescence Compensation Automatic Fluorescence Compensation NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 8 Prime Fluidic System after Long-Term Storage Purge Fluidic System before Shipment Calibrate NovoCyte Fluidics Station II Clean the Optical Filters Clean Fluidic System Troubleshooting Version History Appendix A: NovoCyte Quanteon Laser and Detection Channel Configuration 139 Laser Configuration NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 9 Contents Detection Channel Configuration Appendix B: NovoCyte Quanteon Technical Specifications NovoCyte Quanteon Technical Specifications NovoCyte Workstation and Monitor Specifications NovoCyte Fluidics Station II Specifications NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 10 Contents This page is intentionally left blank. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 11: Prologue

    Agilent regulatory compliance statement and technical support information. Using this Guide This guide describes the information and procedures to install, operate, maintain, and troubleshoot NovoCyte Quanteon flow cytometer and associated standard accessories. For additional information about NovoExpress software, please refer to NovoExpress Software Guide.
  • Page 12 CISPR 11/EN 55011: Group 1, Class A • IEC/EN 61326-1 This ISM device complies with Canadian ICES-001 ISM 1-A ISM Class A Group 1 This symbol indicates the product conforms to following Product Statement standards: NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 13 This symbol indicates that the operation could potentially cause exposure to laser radiation. Protective measures should be taken during the operation. MOVING PARTS This label indicates the moving parts in the instrument. Be careful during the operation. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 14: Glossary

    Band Pass Brilliant Ultraviolet Brilliant Violet BXXX 488 nm (blue) laser excited fluorescence channels or photodetectors. For example, B572 denotes the 572/28 channel excited by the 488 nm laser, and so on. Comma-Separated Values NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 15 637 nm or 640 nm (red) laser excited fluorescence channels or photodetectors. For example, R695 denotes the 695/40 channel excited by the 637 nm or 640 nm laser, and so on. Standard Deviation Sample Injection Probe NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 16: Safety And Limitations

    Safety and Limitations Agilent flow cytometers and associated accessories are limited to be operated by professionals trained by Agilent Technologies or an authorized representative. Agilent flow cytometer incorporates safety measures to protect the operator. Please refer to this guide to operate the instrument in strict accordance with the instructions and in accordance with the provisions of instrument maintenance and repair.

  • Page 17: General Safety

    Agilent Technologies assumes no liability for the customer's failure to comply with these requirements.
  • Page 18 To avoid burns, do not touch the fan guards on the back of the instrument. WARNING The fan guards may get hot during and after instrument operation. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 19: Ergonomics

    Technologies professionals install the instrument, moving the instrument is not recommended. If you need to move the instrument, be sure to contact Agilent Technologies professionals. Agilent Technologies professionals will re-install the instrument in accordance with requirements and ensure the optimal operation of the instrument.

  • Page 20: Electrical Safety

    If the power cord becomes frayed, broken, or damaged, please contact Agilent • Technologies. Do not replace with a lower rated cord. If the equipment is been used in a manner which is not specified by Agilent Technologies, the protection provide by the equipment may be impaired. Laser Safety Laser or laser systems emit intense, coherent electromagnetic radiation that has the potential of causing irreparable damage to human skin and eyes.

  • Page 21: Laser Product Classification

    Biosafety All biological specimens and materials in contact with biological specimens WARNING are potentially biohazardous. In order to avoid exposure to biohazardous material, be sure to follow the guidelines listed below. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 22: Limitations

    If the workstation is used with a network connection, buyers/users are responsible for installation and maintenance of up-to-date virus protection software. Agilent Technologies does not provide warranty with respect to the workstation remaining virus free after installation. Agilent Technologies is not...

  • Page 23: Agilent Regulatory Compliance Statement

    EN standards to which the product was tested to demonstrate compliance. Electromagnetic Compatibility This product conforms to the following regulations on Electromagnetic Compatibility (EMC) and Radio Frequency Interference (RFI): NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 24 Consult your equipment dealer, Agilent Technologies, or an experienced technician for assistance. Changes or modifications not expressly approved by Agilent Technologies could void the user’s authority to operate the equipment. Class B equipment is equipment suitable for use in domestic establishments and in establishments directly connected to a low voltage power supply network which supplies buildings used for domestic purposes.

  • Page 25: Emc Declaration For South Korea

    Schalldruckpegel LP < 60 dB(A) nach DIN EN ISO 7779. Waste Electrical and Electronic Equipment (WEEE) Directive This product complies with the European WEEE Directive marking requirements. The affixed label indicates that you must not discard this electrical/electronic product in domestic household waste. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 26: Regulatory Compliance Identification Number

    For support within the US, please call 800-227-9770. For support within China, please call 800-820-3278 (Desk) or 400-820-3278 (Cell). For users in other countries or regions, contact your local Agilent Technologies representatives or distributors, which may be found at https://www.agilent.com.
  • Page 27 Agilent flow cytometer. User can also attach any other files using this function. Please refer NovoExpress Software Guide for detailed information on this function. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 28 Prologue This page is intentionally left blank. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 29: Introduction

    Introduction Introduction This chapter provides overview of NovoCyte Quanteon flow cytometer, including basic instrument operation principle, system requirements, and system components. Overview Agilent flow cytometer can be used for scientific research, including but not limited to detection and analysis of cell surface antigens, intracellular activity analysis, detection and analysis of apoptosis, DNA content detection, cell cycle analysis of the nucleus, analysis of cell proliferation, and GFP reporter gene detection, etc.
  • Page 30 Incident light typically enters the filter at an angle of 45°. The transmitted light travels in the same direction as the incident light (λ2), whereas the reflected light is reflected in a 90° angle relative to the incident light (λ1) (Figure 1). NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 31 Longpass (LP) filters pass wavelength longer than the cutoff wavelength. For example, a 505 nm LP filter allows wavelengths longer than 505 nm to pass through it and either absorbs or reflects wavelengths shorter than 505 nm as shown in Figure 3. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 32 For example, a 472 nm shortpass filter allows wavelengths shorter than 472 nm to pass through it and either absorbs or reflects wavelengths longer than 472 nm as shown in Figure 4. Figure 4. 472 nm Shortpass Filter Bandpass filters • NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 33 Forward Scatter (FSC). Light that scatters perpendicular to the laser beam is called Side Scatter (SSC). FSC and SSC are related to certain physical properties of cells: FSC - indicates relative size of the cells or particles • NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 34 (525 nm), while other fluorochromes such as PerCP exhibit a larger Stokes shift, absorbing blue light (488 nm) and emitting red light (675 nm). The emission spectra for some commonly used fluorochromes are shown in Figure 7. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 35 (Figure 8). This is known as fluorescence spillover. Spillover can be corrected mathematically by using a method called compensation. Figure 8. Spillover from the FITC Fluorochrome to the PE Detector NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 36 Once fluorescence compensation has been set using compensation control samples, such compensation settings remain valid for any subsequent sample, because compensation subtracts a percentage of the fluorescence signal, independent of the overall fluorescence intensity. Figure 11 illustrates this NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 37: Fluidics System

    NovoExpress Software Guide for details. Fluidics System Agilent flow cytometer fluidics system consists of sample injection probe (SIP), SIP cleaning apparatus, driving pumps, flow cell, solenoid valves, and a series of tubings and connectors. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 38 Sampling box in the Cytometer Control panel from the NovoExpress software. The sample injection probe may perform multiple aspirations for one CAUTION sampling. Do not remove or change the sample tube until the entire sampling process has completed. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 39 When the sheath flow rate remains constant, the lower the sample flow rate, the smaller the core diameter, as illustrated in Figure 13. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 40 The sample flow rate can also be continuously adjusted in NovoExpress software from 5 μL/min to 120 μL/min, which corresponds to a core diameter of 4.6 μm to 22.7 μm. The sheath flow rate is fixed at 6.5 mL/min. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 41: Electronics System

    The peak intensity, or height of the pulse, is measured at this point. As the particle leaves the beam, the pulse trails off below the threshold. Figure 14. Illustration of Pulse Generation NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 42 Each pulse signal is divided into 16,777,216 (2²⁴) levels (Figure 16). The Height, Area, and Width parameters of all available channels for each processed cell or particle are then transferred to the NovoCyte Workstation for further display and processing by the NovoExpress software. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 43: System Requirements

    Introduction Figure 16. Pulse Digitization System Requirements Software NovoExpress software. Workstation NovoCyte Workstation. Fluidics Station NovoCyte Fluidics Station II. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 44: Compatible Sample Tubes

    Introduction Compatible Sample Tubes NovoCyte Quanteon flow cytometer can be equipped with two types of sample tube holder, one for 12 × 75 mm tubes (standard) and one for 1.5 mL or 2.0 mL tubes (optional), respectively. Instrument Reagents NovoFlow Sheath Fluid •...

  • Page 45: Novocyte Quanteon Instrument

    Introduction NovoCyte Quanteon flow cytometer can also be connected to an optional NOTE NovoSampler Q for automatic collection of multiple samples, and an optional NovoCyte Fluidics Cart equipped with 15 L capacity of NovoFlow sheath fluid and waste containers. Please consult with Agilent sales representative for more details.

  • Page 46: Novocyte Workstation And Monitor

    Figure 19. NovoCyte Quanteon Instrument (Rear View) NovoCyte Workstation and Monitor NovoCyte Quanteon flow cytometer comes with NovoCyte Workstation (i.e. desktop or laptop computer), a monitor and associated accessories. Microsoft Windows operating system, Microsoft Office, and NovoExpress software are preinstalled on NovoCyte Workstation.

  • Page 47: Novocyte Fluidics Station Ii

    Figure 20. NovoCyte Workstation and Monitor NovoCyte Fluidics Station II NovoCyte Quanteon flow cytometer comes with one NovoFlow sheath fluid container (3 L), one NovoRinse solution container (500 mL), one NovoClean solution container (500 mL), and one waste container (3 L) by default (Figure 21).
  • Page 48 NovoRinse < 100 mL; NovoClean < 100 mL), or the waste container is full (> 3 L). Any operation of the instrument is prohibited until there are enough reagents added or the waste container has been emptied. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 49: Novocyte Fluidics Cart (Optional)

    Agilent flow cytometer can also be connected to optional NovoSampler Q for automatic collection of multiple samples from a microtiter plate, or from 12 × 75 mm tubes using an Agilent Flow Cytometer 40 tube rack. For ordering information, NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 50 Introduction please visit www.agilent.com or contact Agilent sales representative. For details about the NovoSampler Q, please refer to the NovoSampler Q Operator’s Guide. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 51: Installation

    Installation Installation This chapter provides information on how to install NovoCyte Quanteon instrument and associated standard accessories, including installation warnings, space and environment requirements, instructions for lifting and carrying, and installation procedures. Installation Warnings Ensure the instrument is powered off and is not connected to the workstation WARNING through the USB cable before installing or relocating the instrument.

  • Page 52: Space Requirements

    A minimum space of 150 cm (W) × 80 cm (D) × 90 cm (H) is required to install the NovoCyte Quanteon instrument and NovoCyte Fluidics Station II. Make sure there is at least 20 cm distance between the NovoCyte Quanteon instrument and the adjacent object (i.e. walls). Leave at least 10 cm distance between the NovoCyte Quanteon instrument and the NovoCyte Fluidics Station II.

  • Page 53: Install Novocyte Quanteon

    When NovoSampler Q or NovoCyte Fluidics Cart is purchased, user can refer to associated operator's guide for associated installation procedures. Figure 23 illustrates the typical connection of the NovoCyte Quanteon flow cytometer system. Please follow the instructions below to install each component.

  • Page 54: Connect Novocyte Workstation

    USB port of the NovoCyte workstation (Connection D in Figure 23). There are four communication ports at the back of NovoCyte Quanteon instrument NOTE (see Figure 24). COM 1 port connects to the NovoSampler Q (optional). COM 1 port connects to the NovoSampler Q (optional).

  • Page 55: Connect Novocyte Fluidics Cart (Optional)

    Figure 25. Interface of the Fluidics Ports The reagent containers are connected through the same color coded tubing to the ports at the back of the NovoCyte Quanteon instrument, as shown in Figure 26. Figure 26. Tubing Connections of the Reagent Containers...

  • Page 56: Connect Instrument To Power Supply

    Installation Connect Instrument to Power Supply Connect the NovoCyte Quanteon instrument to a power supply (100-240 VAC, 50- 60Hz) by connecting one end of the supplied power cord to the power port on the instrument (Connection G in Figure 23), and connect the other end of the power cord to the power supply.

  • Page 57: Instrument Operation

    Power on the Instrument Gently press the power switch on the front panel of the NovoCyte Quanteon flow cytometer to turn on the instrument. The power switch light should turn green, indicating that the instrument has entered the initialization process.

  • Page 58: Turn On Workstation

    If the instrument is turned on but you observed the black icon in the NovoExpress status bar, check if there are multiple copies of NovoExpress software opened at the same time. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 59: Add Instrument Reagents

    NovoCyte Fluidics Station II contains pressure sensors that may be CAUTION damaged by spilled liquid. To prevent the risk of sensor damage, do not add reagent while the containers are on the NovoCyte Fluidics Station II. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 60: Empty Waste

    Please make sure the instrument is in Ready status or powered off before CAUTION emptying the waste. Biological samples may be biohazardous. Appropriate personal protective WARNING equipment should be worn when handling the waste, which should be disposed of in accordance with local safety regulations. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 61: Verify And Modify Instrument Configuration

    NOTE detection channels, bandpass filters or dichroic mirrors may not be included in your NovoCyte Quanteon system. Please consult with your local Agilent sales representatives or distributors for more details. Ensure the instrument is properly connected and powered on. Enable the...
  • Page 62 Gently hold the proper optical filter or dichroic mirror and pull it upward to remove it from the slot. Insert the new optical filter or dichroic mirror back into the slot as shown in the following figure. Close the top cover of the instrument. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 63 Figure 29. Change the Optical Filter or Dichroic Mirror Click Complete. The software will automatically read and check the validity of the new optical configuration. Figure 30. Complete Modifying the Optical Configuration Click OK to confirm the new configuration. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 64 Instrument Operation Figure 31. Confirm the New Optical Configuration If the new optical configuration is invalid, there will be message in Instrument NOTE Configuration window. Figure 32. Dialog Window for Invalid Optical Configuration NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 65 NovoExpress Software Guide. Place the sample tube in the tube holder or NovoSampler Q. Fill in the test information in the pop-up window. Click Next to continue. Figure 35. Fill in the Test Information NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 66 Click Report when the QC test is completed. Figure 36. Adjust the Photodetector Gain Figure 37. Running QC Test 10 Ensure the result shows Pass for all the channels. Click Finish to close the window and complete the optical configuration modification. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 67 Click Repeat Test to repeat the QC test. The photodetector gain will be automatically re-adjusted. Please contact Agilent technical support if the QC test failed for three times in a NOTE row. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 68: Instrument Qc Test

    User can also schedule automatic instrument power on or off at selected time with NOTE defined frequency by clicking Schedule Power On & Off from the Operation panel. Please refer to NovoExpress Software Guide for more details about this function. Turn off the Instrument NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 69 Follow the instructions described in Add Instrument Reagents in this guide to add more reagent to NovoFlow, NovoClean and NovoRinse containers. Follow the instructions described in Empty Waste in this guide to Empty the waste container. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 70 Instrument Operation This page is intentionally left blank. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 71: Running Samples

    Slow sample flow rate until the events reach 5,000 in the Lymphocyte gate. This chapter describes the entire sample collection and data analysis processes. Create Experiment • Define Sample Acquisition Parameters • Set Sample Acquisition Conditions • Run Sample • NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 72: Create Experiment

    The extension name of .ncf indicates that the file is a NovoCyte Quanteon experiment file. Click the Next Sample button on Cytometer Control panel to create a new sample to start the experiment.
  • Page 73 For more information, please refer to the NovoExpress Software Guide. After the above operation, the Experiment Manager will appear as shown in Figure NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 74 In this example, select FSC, SSC, B525, Y586, B667, R667 Height and Area as detection parameters. Click the Alias area in the corresponding fluorescent channels to name the detection channel with the name of the fluorophores. In this example, rename NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 75 After the system has started collecting samples, only the Alias of the parameters NOTE can be edited. Selection or de-selection of parameters is not allowed. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 76: Set Sample Acquisition Conditions

    The set of parameters available in the Parameters panel is dependent on the model NOTE of NovoCyte Quanteon flow cytometer. The software will automatically identify the instrument configuration and detect the available channels to be shown in Cytometer Setting > Parameters panel.

  • Page 77: Flow Rate

    Two thresholds can be set at the same time. The primary threshold is based on FSC-H (Height of FSC signal) or SSC-H (Height of SSC signal) or Fluorescent signal Height at available channels, while the second NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 78 Threshold panel for the real experiment. For new experiments, a pre-run helps to optimize the cytometer settings and NOTE determine the most appropriate experimental conditions, including the Stop Condition, Flow Rate, and Threshold. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 79 To detect microsphere with 0.1 µ m diameter, the photodetector gain of SSC needs NOTE to be increased. Please refer to Parameter Settings NovoExpress Software Guide for instructions and more details on adjusting the photodetector gain. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 80: Run Sample

    Information panel becomes T-Lym. To begin collecting this sample, place the CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC sample tube into the sample holder on the NovoCyte Quanteon flow cytometer. Then click the Run button in the Cytometer Control > Experiment Control panel to start sample acquisition (Figure 47). At this time, the sample injection probe will move downward to aspirate the sample.
  • Page 81 Cytometer Control > Experiment Control panel, during the sample acquisition process. After sample acquisition is completed, the NovoCyte Quanteon flow cytometer automatically executes the cleaning process for the fluidics system and the sample injection probe. This helps to reduce cross-contamination between different samples.

  • Page 82: Perform Fluorescence Compensation

    Automatic Fluorescence Compensation Automatic compensation will calculate the fluorescence compensation matrix by the NovoExpress software automatically based on the results of an unstained NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 83 Running Samples control sample and a series of single-stained samples. The compensation matrix can be generated either from samples acquired on NovoCyte Quanteon instrument or from data imported from FCS files. Please refer to Automatic Fluorescence Compensation from Imported FCS Files in this guide for instructions on generate compensation matrix from imported FCS files.
  • Page 84 Manager under the experiment file node. This Compensation Specimen contains a series of single-stained samples as selected in the Compensation Channels (Figure 51). Figure 49. New Auto Compensation Menu Figure 50. New Auto Compensation Window NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 85 The Compensation Channels shown in the New Auto Compensation window will NOTE be all the available channels based on the model of the NovoCyte Quanteon flow cytometer. The NovoExpress software automatically obtains such available channel information from the hardware. Correctly select the Compensation Channels to ensure the correct automatic compensation results.
  • Page 86 (Mean or Median) of the negative and positive populations. The font color of Compensation under the Compensation Specimen node in the Experiment Manager turns blue to indicate the compensation matrix has non-zero NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 87 Figure 55. Spillover Matrix Generated by Auto Compensation Apply Compensation Matrix to Experimental Samples There are two ways to apply the automatically generated compensation matrix to the experimental samples. The first method is to copy and paste the NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 88 The compensation matrix generated by automatic compensation cannot be NOTE modified. However, after it is applied to the experimental samples, you can adjust the compensation matrix or spillover matrix if needed. Figure 56. Copy/Paste the Compensation Matrix NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 89 New Auto Compensation in the Experiment Manager window. Figure 57. Open the New Autocompensation Window Select the single file or multiple files while holding the Shift key in the keyboard. Click Open to import the selected file(s) to the software. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 90 (i.e. Area or Height, Median or Mean). Ensure the correct channel parameter is selected for each single stained sample. To select different single stained sample file, click the file name, select the desired file from the pull-down menu. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 91 FCS file for each fluorescence detection channel. Figure 60. Set the Autocompensation Parameters Click OK and NovoExpress will automatically calculate the compensation matrix based on the samples imported. Once generated, the compensation matrix can be applied to other experiment samples. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 92: Manual Fluorescence Compensation

    PE/PerCP plot and PE spillover into the APC channel on the PE/APC plot. Similarly, for the FITC, PerCP and APC single-stained samples, follow the same procedure to subtract the corresponding fluorescence spillover into the other detection channels. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 93 In the same way, compensate the PE spillover into the PerCP and APC channels on the PE/PerCP and PE/APC density plots. Compensation for PE signal is then completed, and results are shown in Figure 63. The spillover matrix for PE single- stained sample is shown in Figure 62. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 94 Click the Expand Statistics icon in the bottom-right corner of the plot to show the statistical calculation results. Look at the corresponding Mean or Median value to judge if the correct compensation has been achieved. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 95 (Figure 67). Check to make sure the compensation has been applied to the experimental sample and confirm the correctness of the compensation. This compensation matrix can then be used as NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 96 Figure 67. Incorporate the Spillover Coefficients from Single-Stained Samples into the Spillover Matrix Figure 68. Results of T-Lym Sample: Before Compensation Figure 69. Results of T-Lym Sample: After Compensation Please refer to NovoExpress Software Guide for details about the compensation NOTE functions. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 97: Analyze Data

    CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC stained human peripheral blood sample as an example. The general methods apply to the other data analysis as well. Set Sample as Active Sample • NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 98 CD45 PerCP-H. To do so, right-click the FSC-H on the plot and select Height > CD45 PerCP-H from the drop-down list. The X-axis parameter will change to CD45 PerCP-H accordingly (Figure 72). NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 99 Running Samples Figure 72. CD45 PerCP-H/SSC-H Density Plot The NovoCyte Quanteon flow cytometer has a very large dynamic range (0 to 2²⁴=16,777,216), so the data will often only occupy a small area of the plot when using the full-scale axes. Zoom-in to enlarge the data area of interest. To zoom in,...
  • Page 100 Figure 74. Right-click any place on the statistical table to select additional statistical parameters to be calculated for the data, as shown in Figure Figure 74. Statistical Data NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 101: Append Data

    Click Run button in the Cytometer Control panel. If the sample already contains data, a dialog box will pop up. Click the Append button to add more data to the sample. Click the Overwrite button to delete the existing data and re-collect the data. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 102: Delete Data

    Tables, etc., must be saved manually. To save, click File > Save from the menu or click the Save icon . Otherwise, when closing the NovoExpress software, a message will prompt to remind users about saving the data. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 103: Import And Export Files

    (Figure 78). Once enabled, click Export Setting to open the Export Events window. Export settings, including generated file format (i.e. FCS 3.0, FCS3.1 or CSV), can be defined in this window (Figure 79). NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 104 Running Samples Figure 78. Enable the Automatic Data-Exporting Function Figure 79. Configure Exporting Settings NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 105 The NovoExpress software provides powerful data analysis functions, such as data NOTE analysis templates, batch report, statistical tables, cell cycle analysis, cell proliferation analysis etc. For details about the data analysis function of NovoExpress software, please refer to the NovoExpress Software Guide. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 106 Running Samples This page is intentionally left blank. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 107: System Maintenance

    System Maintenance System Maintenance This chapter describes the procedures to maintain NovoCyte Quanteon flow cytometer, including scheduled and unscheduled maintenance. It is important to conduct the instrument maintenance on a regular basis in order to achieve the optimal performance for the instrument and obtain reliable data.

  • Page 108: Preventative Maintenance

    Figure 81. Message in NovoExpress Software when Shutting down The instrument will shut down directly if Clean sample injection probe option is not NOTE selected. Follow the instruction in the software to remove the sample tube from the tube holder. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 109 Prepare a cotton swab or a damp soft cloth with NovoClean solution (1X) and gently wipe the outer surface of the sample injection probe. Figure 83. Wipe the Outer Surface of the Sample Injection Probe NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 110 Figure 84. Software Message Instructing the Cleaning Procedures Place a tube of Milli-Q or 0.2 µ m filtered DI water or NovoRinse solution (1X) in the tube holder. The instrument will shut down automatically after clicking OK button. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 111 Figure 86. Wipe the SIP Cleaning Apparatus’ Lower Surface Prepare a damp cloth soaked with Milli-Q or 0.2 µ m filtered DI water. Wipe the SIP cleaning apparatus back and forth at least five times. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 112 70% ethanol. Empty the reagent container. Fill the NovoFlow container with at least 300 mL of NovoClean solution (1X) and cap the bottle. Fill the NovoClean and NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 113: Replacing Fluidic System Consumables

    Click Replace to continue. Clicking the Close button will cancel the process and allow continued use of the system. However, the consumables should be replaced promptly. Figure 88. Software Message Indicating Fluidic System Consumables Reached Working Capacity NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 114 Click the Replace button and a dialog box in prompted window below. Figure 91. Software Message for Fluidics Consumables Replacement The following fluidic system consumables should be replaced at the same time: NovoFlow sheath fluid in-line filter (1pcs) • Waste filter (1pcs) • NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 115 Figure 93. Software Message to Confirm the Fluidics Consumables are Replaced After confirmation of fluidics consumables being replaced, the NovoExpress software will prompt a window to remind users to conduct the Priming procedure. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 116 Figure 95. Software Message Indicating the Progress of Priming The Priming procedure can also be manually initiated by clicking Instrument > NOTE Fluidics Maintenance > Priming on the NovoExpress software when the instrument is in Ready status. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 117 Replace the NovoFlow Sheath Fluid In-Line Filter Make sure the instrument is in Ready or shut down status. Open the front panel of the instrument. Locate the sheath fluid in-line filter as shown below. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 118 Observe the sheath fluid flows through the filter properly and make sure there is no leakage through the filter-tubing connections. Then run the Priming process when start-up process is completed and instrument is ready. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 119 Replace the old filter with a new one and correctly connect the tubing to the filter. Ensure the side of the filter labeled with "Inlet" is connected to the inlet tubing as shown below. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 120 Replace NovoFlow Sheath Fluid Inlet Filter NovoFlow sheath fluid inlet filter is placed inside the NovoFlow container to filter out micro-particles or impurities in the sheath fluid. Make sure the instrument is in Ready or shut down status. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 121 NovoClean containers, respectively, to filter out micro-particles or impurities in the solution. Make sure the instrument is in Ready or shut down status. Press the metal clip on the quick coupler to disconnect the tubing from the NovoRinse or NovoClean container' cap. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 122: Replace 0.05 Μ M Sheath Fluid Ultrafiltration (Uf) Filter (Optional)

    Replace 0.05 µ m Sheath Fluid Ultrafiltration (UF) Filter (Optional) Agilent flow cytometer 0.05 µ m Sheath Fluid Ultrafiltration Filter is an optional accessory of NovoCyte Quanteon flow cytometer for sterilizing and clarifying biological samples used on the instrument. It has 0.05 µ m pore size which can reduce the background for small particles detection during sample acquisition.
  • Page 123 Ensure the filter is vertically mounted and all the tubing is not twisted. Under some situation when the Ultrafiltration Filter needs to be removed after initial NOTE installation, disconnect the filter from the Luer connectors and connect the male Luer connector directly to the female one. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 124 120 μL/min. Click the Run button in Experiment Control panel to start the sample acquisition. This step is to flush sample tubing. Close the front panel. The installation is completed, and the instrument is ready to use. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 125: Unscheduled Maintenance

    In this case, the following procedure should be conducted to remove the bubbles. When the instrument is in Ready status, click Instrument > Fluidics Maintenance > Debubble from the NovoExpress software to execute the Debubble procedure. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 126: Clean The External Surface Of The Instrument

    The Decontamination function is only available when Decontaminate Instrument NOTE privilege is enabled. Refer to NovoExpress Software Guide for instructions to enable this privilege. Click Instrument > Fluidics Maintenance > Decontamination from the NovoExpress software. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 127: Prime Fluidic System Before Long-Term Storage

    If there is no plan to run the instrument for more than two weeks, a thorough cleaning process is recommended to prevent salt crystallization in the fluidic system. When the instrument is in Ready status, replace all the instrument reagents with Milli-Q or 0.2 µ m filtered DI water. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 128: Prime Fluidic System After Long-Term Storage

    Purge Fluidic System before Shipment If for any reason the instrument needs to be shipped or relocated, perform the following procedure to purge the fluidic system before packaging and shipping NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 129 Pack the instrument and accessories properly for shipment. If there are operating questions when purging the fluidics system, in order to avoid damaging the instrument, please stop purging and contact your local CAUTION Agilent representative or distributor. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 130: Calibrate Novocyte Fluidics Station Ii

    The software will prompt a dialog box indicating the fluidics station calibration is successful. Follow the instructions to place the NovoFlow, NovoRinse, NovoClean, and waste containers back into the fluidics station. Click the OK button. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 131: Clean The Optical Filters

    Repeat Step 3-5 to clean the other side of the filter. Insert the cleaned filter back into the filter slot according to the correct orientation and push it all the way in (Figure 29). Close the top cover of the instrument. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 132: Clean Fluidic System

    When the instrument is in Ready status, click Instrument > Fluidics Maintenance > Cleaning from the NovoExpress software to execute the Cleaning procedure. Figure 113. Cleaning Function in NovoExpress NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 133: Troubleshooting

    Technical Support in this guide. NOTE Table 11 provides guidance on troubleshooting NovoCyte Quanteon instrument and associated accessories. For complete list of NovoExpress software prompted error or warning messages when operating Agilent flow cytometer and associated accessories, please refer to NovoExpress Software Guide.
  • Page 134 Sample flow rate is too high Reduce the sample flow rate. • NovoExpress Workstation is in hibernation Restart NovoExpress Software. • displays Instrument causing the interruption of Restart workstation. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 135 Sampling Pump the sampling pump Leakage from other Incorrect pump or valve or Turn off the instrument and contact Agilent components of the tubing connection or bad technical support for assistance. instrument sheath in-line filter NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 136 Troubleshooting This page is intentionally left blank. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 137: Version History

    Version History Version History Table 12 History of updates Date/Version Changed by Description 08/31/2018 Initial Release 10/31/2019 Second Edition 03/31/2021 Third Edition NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 138 Version History This page is intentionally left blank. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 139: Appendix A: Novocyte Quanteon Laser And Detection Channel Configuration

    PE, PE-Texas Red, PE-Cy5, PE-Cy5.5, PE-Alexa Fluor 700, PE-Cy7 APC, Alexa Fluor 680, Alexa Fluor 700, APC-Cy7 Detection Channel Configuration Table 14 lists the optical filters used in the NovoCyte Quanteon System standard configurations and the recommended fluorochromes for each detector. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 140 NovoCyte Quanteon Laser and Detection Channel Configuration Table 14 Dichroic Mirrors and Bandpass Filters for NovoCyte Quanteon System Excitation Detector Dichroic Mirror Bandpass Typical Fluorochrome Laser Filter 561 nm Forward Scatter (FSC) - 561/14 Side Scatter (SSC) 552 LP 561/14...
  • Page 141 757 LP 780/60 APC-Cy7 Certain fluorescence detection channels, bandpass filters or dichroic mirrors may not be included in your NovoCyte Quanteon system. Please consult with your local Agilent sales representatives or distributors for more details. NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 142 NovoCyte Quanteon Laser and Detection Channel Configuration This page is intentionally left blank. NovoCyte Quanteon Flow Cytometer Operator's Guide...

  • Page 143: Appendix B: Novocyte Quanteon Technical Specifications

    NovoCyte Quanteon Technical Specifications Appendix B: NovoCyte Quanteon Technical Specifications This chapter provides technical specifications of NovoCyte Quanteon flow cytometer and associated standard accessories. NovoCyte Quanteon Technical Specifications Table 15 NovoCyte Quanteon Technical Specifications Optics Laser Solid state laser with on-board thermal-electric...
  • Page 144 NovoCyte Quanteon Technical Specifications Table 15 NovoCyte Quanteon Technical Specifications Sample Flow Rate 5-120 µ L/min, continuously adjustable Average Sheath Flow Rate 6.5 mL/min Sample Aspiration Volume 5 µ L -5 mL Sample Recovery Available Compatibility to Autosampler No fluidic tubing disassembly or re-connection required.
  • Page 145 NovoCyte Quanteon Technical Specifications Table 15 NovoCyte Quanteon Technical Specifications User Management Administrative creation of individual user accounts and user groups with privilege control. Configurable roles for individual users. User operation time tracking. Quality Control Instrument Daily QC Automatic daily quality control with Agilent Flow Cytometer QC Particles.

  • Page 146: Novocyte Workstation And Monitor Specifications

    11.4 × 9.8 × 16.5 in (29.0 × 25.0 × 42.0 cm) (W × D × H) Weight 14.1 lb (6.4 kg) NovoFlow sheath fluid container NovoClean solution container 500 mL NovoRinse solution container 500 mL Waste container NovoCyte Quanteon Flow Cytometer Operator's Guide...
  • Page 148 Running Samples • System Maintenance • Troubleshooting • Version History • Appendix A: NovoCyte • Quanteon Laser and Detection Channel Configuration Appendix B: NovoCyte • Quanteon Technical Specifications www.agilent.com © Agilent Technologies, Inc. 2021 Third Edition, March 2021 *150135* 150135...

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