Luminex Amnis FlowSight User manual

Amnis®FlowSight®Imaging Flow
Cytometer User Manual
For Research Use Only. Not for use in diagnostic
procedures.
780-01286-03 Rev D
February 2020

Technical Support
Telephone: [phone]
North America Toll Free: 1-877-785-2323
International Toll Free: + 800-2939-4959
Email: [e-mail]
www.luminexcorp.com
Luminex Corporation
12212 Technology Blvd.
Austin, Texas 78727
U.S.A.

Table of Contents
Chapter 1: General Information and Safety 1
Chapter 2: Experimental Design 6
Chapter 3: Using the Amnis® FlowSight® Imaging Flow Cytometer 8
Reagents 15
Daily Startup and Calibration 16
Data Acquisition 19
Setting up the Work Area 27
Quick Start Guide to FlowSight® System Operation 30
AutoSampler option 32
Menus 39
Chapter 4: Troubleshooting 42
For Research Use Only. Not for use in diagnostic procedures. ii

Chapter 1: General Information and Safety
This section covers safety information for operating the Amnis® FlowSight® Imaging Flow Cytometer. Anyone who oper-
ates the FlowSight System should be familiar with this safety information. Keep this information readily available for all
users.
The FlowSight Imaging Flow Cytometer is manufactured by Luminex Corporation and has a rated voltage of 100–240
VAC, a rated frequency of 50/60 Hz, and a rated current of 1.5 A.
lEnvironmental conditions: This instrument was designed for indoor use at an altitude of less than 2000 m; at a
temperature from 5°C to 25°C; and at a maximum relative humidity of 80%, non-condensing. During instru-
ment operation the ambient temperature should be maintained within +/- 2 °C. The mains power supply may
not fluctuate more than +/– 10% and must meet transient over voltage category (II). The instrument is eval-
uated to Pollution Degree 2.
lNoise level: The noise level of the FlowSight System is less than 70 dB(A).
lWeight: 56 kg.
lVentilation: Provide at least 3 inches of clearance behind the instrument to maintain proper ventilation.
lDisconnection: To disconnect the instrument from the power supply, remove the plug from the socket outlet—
which must be located in the vicinity of the machine and in view of the operator. Do not position the instrument
so that disconnecting the power cord is difficult. To immediately turn the machine off (should the need arise),
remove the plug from the socket outlet.
lTransportation: The FlowSight System relies on many delicate alignments for proper operation. The machine
may be moved only by a Luminex representative.
lCleaning: Clean spills on the instrument with a mild detergent. Using gloves clean the sample portal and
sample elevator with a 10% bleach solution. Dispose of waste using proper precautions and in accordance
with local regulations.
lPreventative maintenance: The FlowSight System contains no serviceable parts. Only Luminex-trained tech-
nicians are allowed to align the laser beams or otherwise repair or maintain the instrument. The instrument flu-
idic system is automatically sterilized after each day’s use. This reduces the occurrence of clogging. Tubing
and valves are replaced by Luminex service personnel as part of a routine preventive maintenance schedule.
lAccess 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.
lProtection impairment: Using controls or making adjustments other than those specified in this manual can res-
ult in hazardous exposure to laser radiation, in exposure to biohazards, or in injury from the mechanical or elec-
trical components.
lFCC compliance: This equipment has been tested and found to comply with the limits for a Class A digital
device, pursuant to part 15 of the FCC rules. These limits were designed to provide reasonable protection
against harmful interference when the equipment is used in a commercial environment. This equipment gen-
erates, uses, and can radiate radio-frequency energy and, if not installed and used in accordance with the
instruction manual, can cause harmful interference to radio communications. The operation of this equipment
in a residential area is likely to cause harmful interference—in which case the user will be required to correct
the interference at the user’s own expense.
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Amnis®FlowSight®Imaging Flow Cytometer User Manual

Explanation of Symbols
Label Location Hazard
Waste tank Risk of exposure to transmissible
biological disease.
Power supply cover Risk of injury by electric shock.
Power supply Protective earth ground.
Inside rear frame panel Risk of exposure to hazardous
laser radiation.
Interior, side panels near release
mechanisms and next to hood
interlocks
Risk of exposure to hazardous
laser radiation.
On the back of the instrument
No laser radiation is accessible to
the user during normal instrument
operation.
Electrical Safety
Equipment ratings: The FlowSight® System is rated to the following specifications: 100–240 VAC, 50/60 Hz, and 1.5 A.
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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.
Sécurité Electronique
Alimentation: 100–240 V altenatif, 50/60 Hz, 1.5 A.
Les hazards électrique se trouvent dans l’appareil, surtout prés de la source d’alimentation. Pour éviter les choks élec-
triques, introduire la lame le plus large de la fiche dans la borne correspondante de la prise et pousser à fond.
Laser Safety
The FlowSight® System is a Class 1 laser device and complies with the U.S. FDA Center for Devices and Radiological
Health 21 CFR Chapter 1, Subchapter J. No laser radiation is accessible to the user during normal instrument operation.
When the hood is removed, interlocks on the instrument turn the lasers off.
The FlowSight System may have the following lasers:
Wavelength Maximum Power
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
The following laser warning label appears on the interior side panels near release mechanisms.
Using controls, making adjustments, or performing procedures other than those specified in this manual may result in
hazardous exposure to laser radiation.
Sécurité Laser
L'FlowSight c'est une appareil au laser, Classe I, qui se conforme à U.S. FDA Center for Devices and Radiological Health
21 CFR Chapitre 1, subchapitre J. Aucune radiations laser sont accessible a l'utilsateur pendant le fonctionnement nor-
mal. Quand le capot est ouvert, les enclenchements eteindents les lasers.
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Amnis®FlowSight®Imaging Flow Cytometer User Manual

FlowSight peut avoir les lasers suivants:
Longueur
d’opnde
La Puissance Max-
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.
Les etiquettes d’avertissement suivantes sont placeés dans L'Intérieur, de panneaux latéraux pr s de mécanismes de
libération.
Avertissement: L’utilisation des commandes ou les rendement des procedures autres que celle preciseés aux presentes
peuvent provoquer une radioexposition dangereuse.
Biological Safety
Biohazards: The FlowSight® System is rated at BSL1. Do not load or flush samples containing infectious agents without
first exposing the sample to inactivating conditions. It is recommended that samples be fixed in 2% paraformaldehyde
for at least 10 minutes before running the samples on the FlowSight System .
The use, containment and disposal of biologically hazardous materials are required to be in accordance with Personnel
Protective Equipment Directive 93/95/E and are the responsibility of the end user. Follow all local, state, and federal bio-
hazard-handling regulations for disposal of the contents of the waste reservoir.
Prevent waste-reservoir overflow by emptying the container when the waste indicator indicates that it is full.
Run the instrument sterilize routine after each day’s use by clicking on the Shutdown icon.
NOTE: This procedure has not been proven to result in microbial sterility.
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Amnis®FlowSight®Imaging Flow Cytometer User Manual

Sécurité Biologique
Biorisques: L'FlowSight est évalué à un niveau de sécurité biologique L1. Ne pas acquérir ou vider des échantillons conte-
nant des agents infectieux sans les avoir inactivés. Il est recommandé que les échantillons soient fixés dans du para-
formaldéhyde 2% pendant au moins 10 minutes avant d'acquérir des échantillons avec l’FlowSight.
L'utilisation, le confinement et l'élimination des matériels biologiques dangereux sont tenus d'tre en conformité avec les
normes de sécurité relatives au laboratoire et de la directive 93/95/E et restent sous la responsabilité de l'utilisateur.
Respectez la réglementation en vigueur pour le traitement et l'élimination des déchets dans des réservoirs prévus à cet
effet.
Prévenir l'accumulation des déchets en vidant le réservoir lorsque l'indicateur indique qu'il est plein. Stériliser les ins-
truments de routine apre chaque journée d'utilisation. Notez que cette procédure ne garantit pas la stérilité vis à vis des
microbes. 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.
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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.
lChoice of Cell Type: The cell/particle size should be less than 60 microns in diameter.
lFinal Sample Concentration and Volume: At least 1 million cells in 50 μl (2x107 cells/mL) in PBS/2%FBS in a 1.5
mL siliconized microcentrifuge tube.
lProtocols: In general, any established labeling protocol used for flow cytometry will work with the FlowSight
System (see Current Protocols in Cytometry for general labeling techniques). Stain cells on ice in the presence
of azide when possible to reduce non-specific capping of antibody. Use polypropylene tubes, preferably sil-
iconized, to process samples.
lChoice of Fluorochromes: Choose fluorochromes that are excited by the lasers in your FlowSight System (405,
488 and 642 nm are most common). Use the chart below or look online for a spectra viewer that will help you
plan which dyes will work best.
lCompensation: Have a sample of cells each labeled with a single-color for each fluorochrome used (i.e., FITC
only cells, PE only cells, etc.)
lCell Aggregation: Minimize aggregation problems by straining the sample through a 70 micron nylon mesh
strainer or by using an anti-clumping buffer such as EDTA or Accumax™prior to fixation.
lFixation: If fixation is desired, thoroughly fix cells with 1% formalin on ice for 20 minutes.
lNumber of samples: No more than 30 total for feasibility experiments. Please limit the samples to the fol-
lowing; Positive and Negative biologic controls, compensation controls, and experiment samples.
lBrightness of Stain and Stain Balancing: Quantifying the location and distribution of signals in an image is a
demanding task that requires optimized labeling.
Here are a few suggestions to help design the experiment:
lTry to achieve at least a full log shift in fluorescence, as measured by a standard flow cytometer.
lUse the brightest dye for the antigen with the smallest copy number
lThe brightness of probes can be independently controlled by changing the laser power.
However, data quality is enhanced when the brightness levels of all probes excited off a single
laser are balanced to within a log of each other. Probe balancing avoids the saturation of bright
stains when they are combined with dim stains in the same sample.
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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.
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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. Daily oper-
ation involves an initialization of the system and calibration using the Calibration bead reagent, followed by sample runs
and data acquisition, and finally a shutdown procedure which sterilizes the system and prepares for the instrument for
use the following day. Optimizing instrument setup for sample runs is also described in this chapter.
lUser Interface
lReagents
lDaily Calibration and Testing
lInstrument Setup
lData Acquisition
lDaily Shutdown
lAutoSampler
lQuantitative Imaging
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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.
The Image Gallery
Images are displayed in the image gallery during setup and acquisition.
Image Gallery Tools
Icon Name Description
All Select the population to view
Pause Pause/Resume the display
Up/Down Move up or down in the image gallery while paused
Zoom in Enlarges the imagery
Zoom out Resets the zoom
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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
name
Clicking on a channel name will open a window to
select channels collected
The Analysis Area
Graphs are displayed in the analysis area during setup or acquisition. Regions can be drawn on the graphs to create pop-
ulations.
Analysis Area Tools
Icon Name Description
Reset plots Refreshes the graphs with incoming data
Histogram Create a histogram tool
Scatter Plot Create a bivariate scatter plot tool
Pointer Reset cursor to pointer
Line region Draw a line region on a histogram
Rectangle region Draw a rectangular region on a scatterplot
Oval region Draw an oval region on a scatterplot
Polygon region Draw a polygon region on a scatterplot
Select All Selects all plots in analysis area
Tile Tiles the graphs in the analysis area to fill the space
Size Plots Sets size of selected plots to small, medium or large
Populations Opens the population manager
Regions Opens the region manager
Wizards Opens the list of wizards (compensation)
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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.

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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-
name, set the sequence #, choose the data file folder, type the num-
ber of events and choose the population to collect.
Begin Acquisition
Custom Filename Text Type the filename
Seq# Choose the beginning sequence number
Navigate to the folder to save the data
Count Enter the number of events to collect
of Choose the population to collect
Collect a different population while counting the first population.
Click on the bracket to break the link and select population.
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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.
642 nm laser excitation- currently set to ON at 150 mW of power.
SSC laser excitation- currently set to OFF at 10 mW of power.
Brightfield illumination is shown as ON in channels 1 and 9.
Sets the Intensity of the brightfield to 800 counts.
Adjust the speed and sensitivity for the run.
Run fluidics.
Stop fluidics.
Speed and Sensitivity are inversely related.
Focus and Centering can be adjusted using the right and left arrows.
Runs the focus pan test and sets focus automatically.
Runs the startup script and initializes fluidics
Runs the shutdown script and sterilizes the system.
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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
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Amnis®FlowSight®Imaging Flow Cytometer User Manual

Reagents
Sterilizer, Cleanser, and Debubbler
These recommended reagents have been formulated to optimize the performance of the FlowSight® System seals,
valves, syringes, and lines. The use of the recommended reagents is required for proper operation of the instrument.
The Sterilizer, Cleanser, and Debubbler reagents are used in the Sterilize and Debubble scripts.
Reagent Name Source Catalog#
Sterilizer 0.4-0.7% Hypochlorite VWR JT9416-1
Cleanser Coulter Clenz®Beckman Coulter 8546929
Debubbler 70% Isopropanol Fisher Scientific A459
Sheath D-PBS Ca++Mg++ free GE Healthcare Life
Sciences
SH3002803
6506-1L
(10X)
Rinse Milli-Q Deionized water, at least 0.22 µm filtered (use mµ for
µm) Varies 
Calibration
Beads FlowSight Calibration Beads Luminex 400300
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. It is recommended that the waste bottle contain 10% bleach when full.
Sheath Fluid
Two bottles are provided: one labeled Sheath to be filled with phosphate buffered saline (PBS with no surfactants) for
running samples and one labeled Rinse to be filled with de-ionized (DI) water for rinsing the instrument during shut-
down. Fluid is drawn from these bottles into the sheath and flush syringe pumps. The sheath pump helps to control the
speed of the core stream and the size of the core stream diameter. The flush pump is used to clean and flush the sys-
tem.
Fluid Level Warnings
Sensors behind the bottles trigger a pop-up window to alert the user that the volumes are low or the waste volume is
high. The triggers are set such that any script currently running can finish. Do not remove a bottle during the script. Once
the script has finished, remove the bottle and fill the appropriate solution. Waste can be emptied at any time. Dispose of
the waste in accordance with local regulations.
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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
This section describes how to prepare the FlowSight® Instrument for use. The FlowSight Instrument can be left on with
INSPIRE™ launched, but the following instructions also describe how to turn on the FlowSight Instrument if the power is
off.
Turning on the FlowSight® Instrument
1.  Turn on both computers. The large Linux-based computer runs the imaging and the small Windows® computer
runs the user interface. Toggle the power switch on the right lower panel of the instrument from OFF to ON.
2.  Log on with the user name (Amnis) and password (IS100).
3.  Launch the FlowSight® Software using the shortcut on the desktop after the computers and instrument have started.
Preparing to Run Calibration
1.  Fill the rinse bottle with Milli-Q deionized water and the sheath bottle with PBS.
2.  Empty the waste tank. Push on the quick-disconnect buttons to remove the tubing from the waste tank. Add 200 mL
of bleach to the two liter waste bottle. The final bleach concentration for a full waste tank should be 10% for ster-
ilization.
3.  Click Startup. This script fills the system with sheath and flushes out all of the storage rinse that was in the system.
(~12 minutes)
4.  Click Load and place a tube containing 2 drops of FlowSight® System calibration reagent on the uptake port. Vortex
the reagent before use.
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5.  When the beads are running and appear focused and centered, click Start All Calibrations and Tests at the bottom
of the ASSIST view. The calibrations and tests will run and the results saved to the database.
NOTE: Instrument calibrations may also be run individually by selecting a particular procedure under Calibrations
or 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.
NOTE: If adjustments are made to the instrument in order to pass ASSIST, repeat all calibrations and tests to save
the new settings to the database.
Reporting the ASSIST Data
All calibration and test results are saved to a database and can be accessed for reporting and tracking instrument per-
formance.
lTo see the result of the last time a calibration or test was run click on the button to the right of the specific item.
lTo see the result of a calibration or test on a specific date, click on History and choose the item and date in the
drop-down menus.
lTo see a graph of all calibrations and tests for a day, week, month or range of dates, click on Generate Report
and choose the dates.
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Amnis®FlowSight®Imaging Flow Cytometer User Manual

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