Luminex Amnis FlowSight User Manual

Imaging flow cytometer

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Amnis

Cytometer User Manual

For Research Use Only. Not for use in diagnostic

procedures.

780-01286-03 Rev D

February 2020

FlowSight

Imaging Flow

Technical Support

Telephone: 512-381-4397

North America Toll Free: 1-877-785-2323

International Toll Free: + 800-2939-4959

Email: support@luminexcorp.com

www.luminexcorp.com

Luminex Corporation

12212 Technology Blvd.

Austin, Texas 78727

U.S.A.

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Page 1 Telephone: 512-381-4397 North America Toll Free: 1-877-785-2323 International Toll Free: + 800-2939-4959 For Research Use Only. Not for use in diagnostic Email: support@luminexcorp.com procedures. www.luminexcorp.com 780-01286-03 Rev D February 2020 Luminex Corporation 12212 Technology Blvd. Austin, Texas 78727 U.S.A.
Page 2: Table Of Contents
Table of Contents Chapter 1: General Information and Safety Chapter 2: Experimental Design Chapter 3: Using the Amnis® FlowSight® Imaging Flow Cytometer Reagents Daily Startup and Calibration Data Acquisition Setting up the Work Area Quick Start Guide to FlowSight® System Operation AutoSampler option Menus Chapter 4: Troubleshooting...
Page 3: Chapter 1: General Information And Safety
Access to moving parts: The movement of mechanical parts within the instrument can cause injury to fingers and hands. Access to moving parts under the hood of the FlowSight System is intended only for Luminex ser- vice personnel. ...
Page 4 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Explanation of Symbols Label Location Hazard Risk of exposure to transmissible Waste tank biological disease. Power supply cover Risk of injury by electric shock. Power supply Protective earth ground. Risk of exposure to hazardous Inside rear frame panel laser radiation.
Page 5 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Electrical hazards are present in the system, particularly in the main power supply. To protect against electrical shock, you must connect the instrument to a properly grounded receptacle in accordance with the electrical code that is in force in your region.
Page 6 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual FlowSight peut avoir les lasers suivants: Longueur La Puissance Max- d’opnde imale 400-413 nm 150 mW 483-493 nm 60 mW 558-562 nm 50 mW 635-647 nm 150 mW 775-795 nm 90 mW Les etiquettes d’avertissement suivantes sont placeés dans l’interior.
Page 7 Le Niveau de sécurité biologique pour l'instrument est de niveau L1. Spare Parts The instrument contains no serviceable parts. Only Luminex-trained technicians are allowed to repair, maintain, and set up the alignment of the laser beams. For Research Use Only. Not for use in diagnostic procedures.
Page 8: Chapter 2: Experimental Design
® ® Amnis FlowSight Imaging Flow Cytometer User Manual Chapter 2: Experimental Design Sample Preparation Experimental Design ® The FlowSight System can quantify the intensity, specific location, and distribution of signals within tens of thousands of cells per sample. The system can perform most standard flow cytometric assays, and can also leverage the technology's imaging capabilities to discriminate image-based changes within individual cells and cell populations.
Page 9 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual ® ® Amnis FlowSight Imaging Flow Cytometer System Fluorochrome Charts Recommended dyes (based on optimal excitation and detection channels) are in boldface. 1 laser (488) system: Ideal dyes are AF488, PE, PE-TxRed, PE-Cy5, SSC-Ch6, 2 laser (488,642) system: Ideal dyes are AF488, PE, PE-TxRed, SSC-Ch6, and AF647, APC Cy7 3 laser (405,488,642) system: Ideal dyes are AF488, PE, PE-TxRed, SSC-Ch6, and DAPI, AF647, APC Cy7 Band-pass filter values assume 3 laser configuration.
Page 10: Chapter 3: Using The Amnis® Flowsight® Imaging Flow Cytometer
® ® Amnis FlowSight Imaging Flow Cytometer User Manual ® ® Chapter 3: Using the Amnis FlowSight Imaging Flow Cytometer ® ® Operating the Amnis FlowSight Imaging Flow Cytometer ® ™ This chapter describes the operation of the FlowSight System using the INSPIRE for FlowSight Software.
Page 11 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual ® FlowSight Software User Interface The user interface is divided into three areas: 1) the image gallery where channel images are displayed; 2) a work area where graphs of features are displayed; and 3) the controls section where the instrument is controlled. Status inform- ation is displayed along the bottom of the window.
Page 12 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Icon Name Description Settings Adjust the image display Color Turn color ON/OFF Mask Displays the segmentation mask on the images Wrench Tools to measure pixel intensity of displayed images Channel Clicking on a channel name will open a window to name select channels collected The Analysis Area...
Page 13 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual The Instrument Control Panel The instrument control panel provides tools to control instrument operation, data acquisition and status. For Research Use Only. Not for use in diagnostic procedures.
Page 14 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual In the Sample section you can load a sample or return a sample. Sample volume and time remaining is displayed and the rate of the selected population when a sample is running. Loads the sample Returns the sample In the Acquisition Settings section you can type in a custom file-...
Page 15 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual In the Illumination section, you can turn laser and brightfield illu- mination on or off and set intensities. All lasers have variable power and are defined by their excitation bandwidth. 405 nm laser excitation - currently set to OFF and 0 mW of power. 488 nm laser excitation- currently set to ON at 60 mW of power.
Page 16 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual The Task Bar Icon Description Describes the current script Level indicator for pumps Green indicates compensation is being applied to the Intensity feature and images. Yellow- calibrations and tests not run Red- one or more calibrations or tests failed Green- all calibrations and tests have passed For Research Use Only.
Page 17: Reagents
Milli-Q Deionized water, at least 0.22 µm filtered (use mµ for Rinse Varies µm) Calibration FlowSight Calibration Beads Luminex 400300 Beads Waste Fluid ® The two liter waste bottle holds all of the fluids that have been run through the FlowSight System. Add 200 mL of bleach to the empty waste tank.
Page 18: Daily Startup And Calibration
® ® Amnis FlowSight Imaging Flow Cytometer User Manual The left window below is indicating that the Sterilizer solution is low (bright green circle) and needs to be filled. The right window is indicating all bottles are currently OK (dark green circles). Daily Startup and Calibration ®...
Page 19 Tests. Next to each is a green or red rectangle. If the procedure fails, it turns red. If a procedure fails, repeat it. If it fails twice, see Troubleshooting or call your Luminex Field Service Representative. 6. When the calibrations and tests have all passed, close the ASSIST window. This window can be re-opened under the Instrument menu.
Page 20 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual For Research Use Only. Not for use in diagnostic procedures.
Page 21: Data Acquisition
® ® Amnis FlowSight Imaging Flow Cytometer User Manual Data Acquisition ® After the FlowSight System is calibrated, you are ready to acquire experiment data files. The sample is loaded into the sample pump. Sample is injected into the flow cell to form a single core stream that is hydrodynamically focused in front of the imaging objective.
Page 22 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual 3. Turn on each laser used in the experiment by clicking on the wavelength. Observe the scatter plots of Raw Max Pixel for the channels used in the experiment. Set the laser powers so each fluorochrome has Raw Max Pixel Intens- ities between 100 and 4000 counts, as measured in graphs and there is no saturation.
Page 23 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual 6. Create additional graphs if desired to gate on cells of interest. Recommended: Scatterplot of Area versus Aspect Ratio of brightfield to gate on cells and eliminate debris. Scatter plots or histograms of Raw Max Pixel for the channels used in the experiment for setting laser power. Scatterplots or histograms of Intensity for the channels used in the experiment.
Page 24 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual The setup can be saved as a template file (.ist) for future experiments under the File menu. 1. Enter the number of cells you want to acquire in the Count box and choose the population in the drop-down menu. Optional: Click on the bracket to break the link to choose a population to count and a different population to collect.
Page 25 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual 7. To prematurely stop acquisition click . The system prompts you to either discard the acquired data or to save the collected data in a file. The acquisition can be paused and resumed by clicking 8.
Page 26 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual 4. Verify the channel for the sample loaded and click Next. 5. Type a Filename for the compensation file. Choose the Seq# and folder to save the data if not already done. 6.
Page 27 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual 8. Optional: If desired, compensation values can be adjusted by clicking on the compensation tool button in the graph. 9. For example changing the matrix value in the 2x2 submatrix from 0.189 to 0.10 changes the compensation as shown from the graph above (properly compensated) to the graph below (under compensated).
Page 28 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual 11. Click Exit when finished and save the matrix (.ctm) when done. The previous instrument settings will be restored and the compensation matrix is now being applied to the Intensity features and images.
Page 29: Setting Up The Work Area
® ® Amnis FlowSight Imaging Flow Cytometer User Manual 5. To set the display mapping adjust the right and left green bars in the graph. You will adjust the Display Intensity set- tings on the graph (the Y Axis), to the Pixel Intensity (the X axis). The range of pixel intensities is 0-4095 counts. The display range is 0–255.
Page 30 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual ® NOTE: See the IDEAS User Manual for more information on features and graphing. The tools functionality is the ™ same in INSPIRE as in IDEAS. ® Table 1. Features available for FlowSight Name Mask_Channel Definition...
Page 31 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual 4. Confirm your gating by viewing the populations created by gates in the image gallery. 5. Create scatterplots and histograms as required. For Research Use Only. Not for use in diagnostic procedures.
Page 32: Quick Start Guide To Flowsight® System Operation
® ® Amnis FlowSight Imaging Flow Cytometer User Manual Example Template for 4-Color Experiment ® Refer to the IDEAS User Manual for more detailed information on graphing tools, moving regions or panels or managing populations. ® Quick Start Guide to FlowSight System Operation 1.
Page 33 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual 2. Check to be sure the buffer containers are full and the waste tank is empty. 3. Select Startup and the instrument will flush the system and load sheath in ~12min. 4.
Page 34: Autosampler Option
® ® Amnis FlowSight Imaging Flow Cytometer User Manual 16. Shut the system off by pressing the Shutdown button. The system sterilizes in ~40 min. AutoSampler option ® The AutoSampler enables unattended operation of samples in 96 well plates loaded into the FlowSight Instrument.
Page 35 7. At a minimum, each well requires an Output File Path, Max Acquisition Time, and Template File in order to be con- sidered ‘defined’. Optional parameters can be added to the definition in the next step. NOTE: Luminex highly recommends to leave the validate sample option 'Yes'. See notes below regarding validating samples.
Page 36 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual 11. Click Add/Remove Well Parameters to choose the parameters you want to report for the wells. There are several categories of parameters that may be chosen as a group or individually. Select or clear the desired parameters. The user can also define custom parameters.
Page 37 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual 13. You can edit values for some of the Custom and many of the Standard parameters. You can do this for all selected wells or for individual wells. For example, if you want to collect with Max Acquisition Time 10 minutes for the selected wells, type 10 in the ‘Apply to selected’...
Page 38 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Start the AutoSampler 1. Click Start to run the plate. The Auto Sampler Unattended Operation window opens with the Plate Definition you just saved. If you wish to choose a different definition, browse for it by clicking on the folder icon. ...
Page 39 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual For example, here is a run with a failure to detect objects in column 3 for the first 2 rows (A3 and B3 failed). The wells are shown in red and the error status is displayed. An alert email was sent as entered in the plate definition. During a Run ...
Page 40 Brightfield (if the template has BF off then BF must be turned on). This step occurs whether validation is ON or OFF in the plate definition. NOTE: Luminex highly recommends that the validation is 'Yes' in the plate definition for every well. ...
Page 41: Menus
® ® Amnis FlowSight Imaging Flow Cytometer User Manual Menus ™ The menu bar is located in the upper-left portion of the INSPIRE screen. It consists of the following drop-down menus. This section describes the functions that are assessable from the menus ...
Page 42 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Start Camera: Turns on imaging if it has stopped. Calibrate with ASSIST: Opens the Calibrations and Tests window. Load Sheath: Fills the sheath syringe with sheath fluid and an air bubble that facilitates stable flow. ...
Page 43 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Vertical: View the image gallery and analysis area side by side. Horizontal: View the image gallery and analysis area top and bottom. Auto-resize Analysis Area: Adjusts the layout to accomodate graphs and images when the image gal- lery changes size ...
Page 44: Chapter 4: Troubleshooting
Imaging Flow Cytometer User Manual Chapter 4: Troubleshooting This section is designed to help you troubleshoot the operation of the FlowSight ® System. If additional assistance is required, contact the Luminex Technical Support. System see Unstable fluidics (Air or clog in system) ...
Page 45 With the system powered down look for leaking sheath. Verify the fluid lines Fluid lines are leaking mount snuggly into position. Call Luminex Technical Support. The sheath syringe should contain 2-4 mL of air to buffer the movement of Air buffer in the sheath the pump’s microstepper motor.
Page 46 Erroneous fluid level indicator Power down and power up the instrument, if this does not fix the problem, Sensor is broken call Luminex Technical Support. For Research Use Only. Not for use in diagnostic procedures.
Page 47 Calibration and/or test Re-run the test by clicking in the box next to the test, and pressing the start failure button in the popup window. If the test fails three times in a row, call Luminex Technical Support. Focus adjustor cal- Verify brightfield is working properly.
Page 48 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Symptom Possible Causes Recommended Solutions Flow Core Position test See solution above (For more information, see Flow Core Axial Stability test fail- failure ure.). Focus Percentage test See solution above (For more information, see Flow Core Axial Stability test fail- ...
Page 49 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Symptom Possible Causes Recommended Solutions Open the Windows Task Manager by simultaneously pressing ™ <Ctrl + Alt + Del>. Click the Applications tab. If the INSPIRE ® FlowSight Software status is ‘Not Responding’, select the ™...
Page 50 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Symptom Possible Causes Recomended Solutions Camera is not running Click Run/Setup. If the camera is already Click Stop to stop the camera, and then click Run/Setup. running Imaging is paused Click Resume.
Page 51 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Symptom Possible Causes Recomended Solutions The core tracking and focus tracking should not change significantly from Core stream position is day to day. If either value changes radically, objects may rotate due to inter- Objects are rotat- grossly off-center within actions with the sheath.
Page 52 ® ® Amnis FlowSight Imaging Flow Cytometer User Manual Symptom Possible Causes Recomended Solutions Image display settings Increase the image display gain and/or change to log in the appropriate are set too low camera channel. Sample did not label well Look at the sample with a fluorescent microscope.
Page 53 Power down and power up the instrument, if this does not fix the problem, intensity levels variable output call Luminex Technical Support. Intensity set before Allow the system to stabilize after loading a sample, and then click Set desired flow speed has Brightfield intens- Intensity.
Page 54 Luminex Corporation (Luminex) reserves the right to modify its products and services at any time. This guide is subject to change without notice. Although prepared to ensure accuracy, Luminex assumes no liability for errors or omissions, or for any damages resulting from the application or use of this information.