Orflo MOXI GO User manual

For Research Use Only. Not For Use In Diagnostic or Therapeutic Procedures.
Moxi GO™
USER GUIDE

This document is provided to customers who have purchased Orflo Technologies, LLC (“Orflo”) equipment, software,
reagents and consumables to use in the operation of such Orflo equipment, software, reagents, and consumables.
This document is copyright protected and any reproduction of this document in whole or any part is strictly prohibited,
except as Orflo may authorize in writing.
Limitations: The Moxi GO is intended for research use only. It is not intended for use in medical diagnostic or
therapeutic procedures, and it has not been approved or cleared by the Unites States Food and Drug Administration
for such uses.
Restrictions of Use: Software that may be described in this document is furnished under a license grant. Customer
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Disclaimer: The information in this document is subject to change without notice and should not be construed as a
commitment by Orflo. Neither Orflo nor any of its affiliated corporations assumes responsibility for any errors that
may appear in this document. This manual is believed to be complete and accurate at the time of publication. In no
event shall Orflo or any of its affiliated corporations be liable for incidental or consequential damages in connection
with or arising from the use of this manual.
Trademarks: Portions of this document may make reference to other manufacturers and/or their products, which
may contain parts whose names are registered as trademarks and/or function as trademarks of their respective
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manufacturer’ and or their product names as trademarks.
© 2016 E.I. Spectra LLC dba Orflo Technologies. All rights reserved.
Rev. 1.4, 03/2017
Orflo Technologies and Moxi GO™ are trademarks of E.I Spectra LLC dba Orflo Technologies, registered in the U.S.
and other countries.
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registered in the U.S. and other countries.
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FlowJo is a trademark of Treestar, Inc.
Flowmi™ is a registered trademark of SP Scienceware, Division of SP
Accutase®is a registered trademark of Innovative Cell Technologies.
TÜV SÜD and the TÜV SÜD symbol are registered trademarks of TÜV SÜD Aktiengesellschaft.

Contents
Introduction ................................................................................................................................... 1!
About the User Guide ................................................................................................................... 1!
Conventions Used in the User Guide ........................................................................................... 2!
Safety Precautions........................................................................................................................ 2!
Electrical Safety ........................................................................................................................ 2!
Laser Safety .............................................................................................................................. 2!
Biological Safety........................................................................................................................ 3!
Moxi GOTM System Overview ....................................................................................................... 4!
Moxi GOTM Accessories................................................................................................................ 5!
Getting Started.............................................................................................................................. 6!
Using the Moxi GO™.................................................................................................................... 7!
Home Screen ............................................................................................................................ 7!
Settings ..................................................................................................................................... 8!
A number of settings are made available to the user by selecting the Settings icon............. 8!
Date and Time ....................................................................................................................... 8!
Passcode Lock ...................................................................................................................... 8!
Fluorescence Gain ................................................................................................................ 9!
Auto Noise-Find..................................................................................................................... 9!
Acquiring and Analyzing a Sample ......................................................................................... 10!
General Sample Preparation Guidelines and Considerations ............................................. 10!
General Instructions for Running a Test (All Applications) .................................................. 11!
Running the Open Flow Cytometry Application................................................................... 12!
Running a “Cell Cycle” Test................................................................................................. 14!
Analyzing a Sample ................................................................................................................ 18!
Test Screen Outputs............................................................................................................ 18!
Fluorescence vs Size Dot Plot............................................................................................. 18!
Histograms .......................................................................................................................... 19!
Managing the Data.................................................................................................................. 20!
Data Analysis Workflow....................................................................................................... 20!

Re-scaling the Size Range (X-axis)..................................................................................... 21!
Gating the data .................................................................................................................... 21!
Managing Data Files and Storage........................................................................................... 29!
Saving, Naming and Renaming Files .................................................................................. 29!
Saving and Deleting Data/Tests.......................................................................................... 30!
Exporting Data Screenshots................................................................................................ 30!
Editing Saved Tests............................................................................................................. 31!
Compare/Overlay Saved Tests ........................................................................................... 32!
Exporting Data – Connecting Via USB (USB on-the-go) ........................................................ 34!
System Information ................................................................................................................. 35!
Instrument Firmware and Software Upgrades ........................................................................ 35!
Updating the Firmware............................................................................................................ 36!
Moxi GO™ Apps ..................................................................................................................... 37!
Troubleshooting .......................................................................................................................... 38!
Error Messaging ......................................................................................................................... 40!
Maintenance and Storage........................................................................................................... 41!
Specifications for the Moxi GO™ and MFS Cassettes............................................................... 43!
Ordering Information................................................................................................................... 44!
Technical Service ....................................................................................................................... 45!
Warranty ..................................................................................................................................... 45!
Applicable Patents ...................................................................................................................... 46!

Moxi GO™ User Guide
Page 1
Introduction
The Moxi GO™ from Orflo Technologies is a benchtop instrument that utilizes a combination of
a 532 nm laser (MXG001 model) or 488nm laser (MXG002 model), one photomultiplier tube,
Coulter Principle-based cell measurements, and on-board software to provide easy-to- run
applications and data analysis. The 532nm system comes with a built-in 561nm/LP PMT
emission filter, ideal for phycoerythrin (PE) conjugated antibodies, propidium iodide (PI) viability
staining. The 488nm system comes with both a 525/45nm PMT emission filter (ideal for FITC,
GFP and other similar dyes) and a 561nm/LP filter (ideal for PE and PI). The filters are
swappable depending on the sample to be run. The instrument relies on disposable cassettes
for sample handling, which alleviates the need for flow cell cleaning and fluidics maintenance
and the instrument is small enough to be portable between the lab bench and the hood.
The Moxi GO™ enables highly accurate cell counts and quantitative assessments of cell
viability, apoptosis, other labeled antibody markers along with multiplexed bead-based assays
for protein and cellular analysis. Data acquisition occurs within approximately 10 seconds per
test. Test results are displayed in the form of histograms and scatter plots, and quantitatively
show cell number, cell percentage(s) gated, and mean fluorescent intensity value(s) in a
graphical display. The instrument can store up to 16 Mb of data and the data may be
downloaded to a PC or Mac via USB connectivity.
IMPORTANT NOTE:
The Moxi GO™ is intended for research use only. It is not intended for use in medical diagnostic
or therapeutic procedures, and it has not been approved or cleared by the Unites States Food
and Drug Administration for such uses.
About the User Guide
The Moxi GO™ User Guide provides detailed information for operating, maintaining, and
troubleshooting the Moxi GO™, including the system, the Cassettes, and application workflows.
This includes information on how to analyze test results using the onboard analysis features of
the software.
The user guide does not include instructions for analyzing the provided/output flow cytometry
standard (FCS, version 3.1) files offline in third party software.
Similarly, the user guide does not include detailed protocols for preparing samples prior to
running them on the instrument. The results of the assays are dependent upon the proper use
of reagents and the instrument.

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Conventions Used in the User Guide
Within this manual, the following conventions are used to call out areas of safety concern
(warnings and cautions) and important points when running and analyzing experiments.
WARNING Alerts you to a situation that may cause injury to the user.
CAUTION Alerts you to a situation that may cause damage to the system, loss of
data, or incorrect results.
NOTE Indicates additional related information that may be helpful to the user.
Safety Precautions
The Moxi GO™ has integrated safety features that are designed for the protection of the user.
THE INSTRUMENT SHOULD BE USED ONLY BY PROPERLY QUALIFIED AND TRAINED
LABORATORY PERSONNEL, AND SHOULD BE USED ONLY AS DIRECTED IN THIS
GUIDE. PLEASE REVIEW AND UNDERSTAND ALL SAFETY INSTRUCTIONS BELOW, AND
AS INDICATED THROUGHOUT THE USER GUIDE, BEFORE OPERATING THE SYSTEM.
Electrical Safety
WARNING To avoid the danger of electric shock, including the possibility of permanent
injury or death:
•Prior to use, verify that the USB cable and USB charging adapter are plugged
securely into a properly grounded AC power outlet. Verify that the connection
between the USB cable and the instrument is secure and the AC power in your
location is within the specifications for the instrument (see page 43).
•Do not immerse the instrument, USB cable, or USB power adapter in liquid or allow
liquid to enter the instrument.
•Do not attempt to disassemble or service the Moxi GO™. The instrument has no
user serviceable parts. All service must be performed by ORFLO, or an
approved ORFLO vendor.
Laser Safety
WARNING The Moxi GO™ is a Class 1 laser product in accordance with IEC 60825-
1:2014. The system contains a Class 3B (IIIb) laser that operates at 532 nm
(MXG001) or 488nm (MXG002) with a maximum output power of 60 mW.

Moxi GO™ User Guide
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Direct exposure to laser radiation, even for a fraction of a second, can be
harmful to skin, eyes, or other body parts, including posing a risk of burns and
permanent vision impairment or blindness. The following guidelines should
be followed to avoid exposure:
•An interlock on the door prevents the laser from operating when the door is open.
Do not attempt to defeat the interlock and run a test with the door open.
•Do not operate the Moxi GO™ if there is any damage to the instrument’s enclosure.
•Do not attempt to disassemble or service the Moxi GO™ to service or adjust the
laser. The instrument has no user serviceable parts, and attempts to adjust or
service the laser may result in hazardous radiation exposure. All service must be
performed by ORFLO, or an approved ORFLO vendor.
Biological Safety
WARNING Biological samples have the potential to transmit harmful or fatal disease.
The following guidelines should be followed to minimize the risk of exposure
to biohazardous materials.
•Handle all biological samples as if they are capable of transmitting harmful or fatal
infections.
•Wear appropriate personal protective equipment (PPE) including clothing, eyewear,
and gloves.
•Do not pipette by mouth.
•Dispose of biological waste in accordance with all applicable local, state, and
federal regulations.

Moxi GO™ User Guide
Page 4
Moxi GOTM System Overview
The Moxi GO™ Kit includes the Moxi GO™ flow cytometer, USB Cable, USB Power Adapter,
one pack of 25 MF-S+ Cassettes, a Quick Start Reference Guide, and Registration Card. The
following figure shows the main visible components of the Moxi GO™ and the table provides a
description of each component.
Power/Reset Button
USB Cable Port (behind)
Cassette Tray Lever
Cassette Tray Door
Touch
Screen
Display
Disposable Cassette

Moxi GO™ User Guide
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Component Function
Touch Screen Display Allows user to interface with the instrument by pressing
icons. Displays all information needed for operation and
analysis of results.
Power/Reset Button Turns instrument on and off, and resets the unit when
pressed and held for >5 seconds.
Cassette Tray Door Door is manually opened and closed by user to allow
insertion of a cassette into cassette tray.Door must
remain closed to start and while running a test.
Cassette Tray Lever Depress lever to open cassette tray and allow cassette
insertion and removal. Release of lever, with cassette in
place, secures cassette into position.
USB Cable Port Connects instrument to USB cable.
Cassette Disposable cassette used for handling the samples. Each
cassette contains two fill ports, one at each end, allowing
for two tests to be run per cassette.
Moxi GOTM Accessories
The Moxi GO™ uses a USB Cable and Power Adapter for power source. The Moxi GO™ Kit
will come with the power adapter required for your local region. For more information on the
approved regional power adapters, please see Section 14, Ordering Information. Additional
adapter types may be available for other regions, please contact technical support at ORFLO for
more information. The 488nm system (MXG002) also comes with the additional (swappable)
561nm/LP filter
USB Cable Power Adapter (US style)
Part Function
USB Cable Connects instrument to Personal computer (PC or
Mac) and power adapter
Power Adapter, US version Connects USB cable to a 110 VAC outlet
561nm/LP Filter With the 488nm (MXG002) system, the default
525/45nm filter can be swapped with this one for
use with different fluorophores (e.g. PE)

Moxi GO™ User Guide
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CAUTION Use only a ORFLO supplied USB Cable and Power Adapter. Use of other
products may result in inaccurate test results or damage to the
instrument, and will void warranty.
Getting Started
The Moxi GO™ is shipped in a condition ready for initial use, however it is recommended to
update the flow cytometer with the most current software version available upon receipt.
NOTE: Save all packaging as these materials are recommended for use should you
need to send instrument for service. This packaging is designed to prevent shipping damage.
To begin, remove Moxi GO™, USB cable and power adapter from the inner white box. Plug the
mini-USB end of the USB Cable into the instrument. Plug the opposite end of the USB Cable
(standard USB) into the Power Adapter. Plug the Power Adapter into an AC outlet that matches
the rating of the Power Adapter. Charge the Moxi GO™ overnight before first use and to keep
the instrument plugged in to ensure the instrument remains fully charged
NOTE The instrument has an internal, rechargeable battery, which allows the
instrument to power on, and allows users to access the software and files. To power the laser
the instrument must be plugged into an electrical outlet (AC).
NOTE If instrument is less than 50% charged you may not be able to power the laser,
even if plugged in.

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Using the Moxi GO™
Press the Power Button to turn on the instrument. The Home Screen will become available.
Home Screen
NOTE:The home page appearance may change as additional applications are launched
and new icons are added to the Moxi GO software program.

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Settings
A number of settings are made available to the user by selecting the Settings icon.
Date%and%Time%
Set the date and time by pressing the Settings icon on the Home screen of the instrument.
Then use the left and right arrows on the screen to highlight the Date field. Use the up or down
arrows to change and select the Month field value. Use the right and left arrows to toggle
through the Month, Day, Year and Time fields. When completed, select Done.
Passcode%Lock%
The Moxi GO™ can be configured to require a password login to use the instrument and access
stored data. To set the lock code, select the Settings icon and then use the left and right arrows
on the screen to highlight the “Passcode Lock” field. Use up or down arrows to change
“Passcode Lock” field value to “On” and select Done. The system presents a keyboard; enter a
four digit numeric passcode. The user is required to “Re-enter Passcode” to prevent accidental
entry of a wrong code. Upon successful re-entry, the system displays a “lock enabled” message
and will require a passcode for access on startup and after entering sleep mode.
CAUTION:If you forget the passcode, the entire system will have to be
reset and data could be lost
In order to turn the Passcode Lock off, enter the correct passcode and then select “off” in the
passcode lock window. Keep the passcode recorded in a safe place to prevent loss of data.

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Fluorescence%Gain%
The Fluorescence Gain field can be set to adjust the level of fluorescence gain for the “Open
Flow Cytometry” assay only. Four possible values (Low, Default, Medium, High) can be entered
here and will seed the value of the dropdown list on the “Open Flow Cytometry” application start
screen. The need to change this setting is uncommon as the Default setting is designed to
properly display the majority of sample preparations. However, for extremely bright samples, it
may be necessary to lower the fluorescence gain to the Low setting to prevent the PMT from
saturating. When a sample is too bright, the user will see points accumulating at the top of the
scatter/dot plot or, in cases of complete saturation, all the point accumulating in a line at the 0
mV level on the scatter plot. To set the fluorescence gain, select the Settings icon and then
use the left and right arrows on the screen to highlight the Fluorescence Gain field. Use up or
down arrows to change and select the field value, and then select Done.
Note: the user can also make a one-time (per-test) change to the fluorescent gain value
of the “Open Flow Cytometry” by touching the value on the test start screen as described
in Step 3 of the “Analyzing a Sample” | “Running a Test” section.
Auto%Noise-Find%
This field sets the behavior of the system with respect to identifying system noise during tests.
There are two possible settings: Yes or No. If set to Yes, the system applies an algorithm to
each test, after acquisition, to try to identify the noise (and small-particle “debris”) in the sample.
If set to No, the system will use a hard-coded values for the noise location for each test, capping
the noise area to a region defined by events <2.9µm AND fluorescence levels <10mV.
To set the Auto Noise-find, select the Settings icon and then use the left and right arrows on
the screen to highlight the Auto Noise-find field. Use up or down arrows to change and select
the field value to Off or On, and then select Done.

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Acquiring and Analyzing a Sample
General%Sample%Preparation%Guidelines%and%Considerations%
There are five key considerations for preparing a sample for analysis with the Moxi GO™:
1. Solution Conductivity – Cells must be suspended in 0.9% salt solution (e.g. PBS or
equivalent) to ensure proper conductivity for unit operation and to ensure accurate
particle sizing. Water, hypotonic, or hypertonic solutions are not acceptable diluents.
Deviations of ~50% of percent salt solution can be tolerated without sacrificing count
performance. However, this will have effect on the reported particle diameter
measurement.
2. Particle Size and Concentration – For best results tests must be run within the size
and concentration range specifications of the cassette, as specified below.
a. Size Range: 3-16 µm effective diameter (14 – 2144 fL volume)
b. Optimal Concentration range for:
i. Cell/Bead Counting: 1 x 104– 1.75 x 106total particles/ml
ii. Fluorescence Detection - Cells:
1. Open Flow - 1 x 105– 5 x 105particles/ml
2. Cellular Assay Kits: Please refer to specific Assay Kit Data sheet
iii. Fluorescence Detection – Beads:
1. Please refer to specific Assay Kit Data sheet
Optimal concentration ranges up may vary slightly depending upon your cell or
bead type.
3. Single Cell Suspensions – While cassettes do have a pre-filter for removing sporadic
large particles, cell samples should be prepared as single cell suspensions to avoid
clogging. Clusters/aggregates can be broken apart with mechanical trituration and/or
protease dissociation (e.g. Accutase®). Larger particles or stubbornly aggregated
clusters can be removed with a 40µm cell strainer. Standard centrifuge tube strainers
can be used for larger volumes or a pipette-tip Flowmi™ strainer for smaller volume
filtering.
4. Fluorescent Labeling/Stains - The Moxi GO™ uses a 532 nm laser (MXG001) or a
488nm laser (MXG002) for excitation. Both configurations come with a 561 nm/LP
detection filter. Dye and fluorophore selections, for that filter should be based
accordingly: Phycoerythrin(PE), Sulfarhodamine, PE/Cy5, JC-1 (aggregates). The
488nm system is also equipped with a 525/45nm filter for use with FITC, GFP and other
dyes with similar excitation/emission spectra.
Materials%Required%

Moxi GO™ User Guide
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•Cell or bead sample, previously prepared according to reagent or assay kit instructions
(diluted and dissociated, if necessary); 75 µL minimum needed per test.
•MFS Cassettes
•Pipette and appropriately sized pipette tips
General%Instructions%for%Running%a%Test%(All%Applications)%
The following represent a hi-level overview of the steps when running any application on the
Moxi GO™.
1. Turn the instrument on by pressing the power button and the Home screen will be
displayed.
NOTE The instrument must be plugged into an electrical outlet (AC) in order to
power the laser and run an application.
2. Select an application by pressing one of the icons displayed on the screen. Basic
instructions, such as loading a cassette, are then presented in the following screens of
the application.
3. Open the Cassette Tray Door and depress the Cassette Tray
Lever. Slide a cassette, with ORFLO lettering-side up, into the
cassette tray (either end 1 or 2). Release the Cassette Tray
Lever and close the Door.
Note Should this result in an error make sure that (a) your
cassette is pushed into the cassette tray completely such that it
locks into place; (b) you are using a new cassette, or unused
side of a cassette.
4. The Moxi GO™ will automatically perform a calibration sequence, as outlined in the
images below, to align the laser to the cassette flow cell. This occurs for all tests except
“Size Histogram”. When complete the system prompts the user to open the Cassette
Tray Door and load sample.
NOTE System status messages and commands, such as “Aligning the laser” are
displayed in the black bar at the top of the screen.
NOTE For the first test run, following power up, an initial system calibration is performed
(“System Calibration” in image above/left). Following that initial test, only the standard
laser alignment (images to above/middle and above/right) is performed.
5. System messages at the top of the screen prompt the user to “Open the Door” (image

Moxi GO™ User Guide
Page 12
below/left) and pipette a 75 µL sample (“Enter the Sample”, image below/middle) into the
fill port of the cassette and close the Door.
CAUTION: The current disk usage space is also indicated, as a
percentage, at the bottom right of the screen at this point. Check how full the
disk is BEFORE loading your sample.
NOTE: The system provides the user a 60 second window to complete
each step (opening the door, loading the sample, waiting for the fluid to reach the
start electrode). If the time limit for any of the steps is exceeded, the test will
automatically cancel and the user will be returned to the Home screen
6. Acquisition begins when the door closes. While the system waits for sample/fluid
detection an “Analyzing…” message is indicated in top left of the screen. Once the fluid
is detected, the system message changes to “Counting…” and the results are
dynamically populated on the dot plot. Acquisition will be complete in approximately 10
seconds for most samples, but may be longer for higher concentration, sticky, or
aggregated samples. If no fluid is detected after 60 seconds the test is auto-cancelled
WARNING Opening the door during analysis will auto-cancel the test. This
protects users from exposure to the laser.
Running%the%Open%Flow%Cytometry%Application%%
The Open Flow Cytometry application is an open-format test that allows the user to design their
own assays using antibodies and dyes. Assays must use fluorophores compatible with the
system configuration:
•MXG001: 532 nm laser excitation and 561nm/LP filter.

Moxi GO™ User Guide
Page 13
•MXG002: 488nm laser with 561nm/LP or 525/45nm filters
It might be necessary to perform reagent or cell titration assays and/or adjust the Fluorescent
Gain to achieve optimized test results.
The general process for running an Open Flow Cytometry assay is outlined in the following
steps; these refer to the corresponding image sequence below.
1. As with all assays, please follow the sample preparation guidelines above (“General
Sample Preparation Guidelines and Considerations”). The recommended volume
required for a test is 75 µL.
2. Select the “Open Flow Cytometry” application from the Home screen (image
below/left).
3. For the “Open Flow Cytometry” application, an option of selecting a fluorescence
gain is available (image below/right). To adjust the fluorescent gain for an individual
acquisition, select the gray box next to the “Fluorescent Gain” field. Four options, in
order of increasing gain, are displayed: Low, Default, Medium, and High. The initial
value populated in this field is determined by the global “Fluorescence Gain” setting
(set from the Settings icon on the Home Screen).
4. A pop-up menu will appear with the available options (image below/right). Touch the
desired value to change the setting.

Moxi GO™ User Guide
Page 14
5. Select “Start” or insert a new cassette and close the door to begin sample
acquisition. See “General Instructions for Running a Test” section for further detail.
Running%a%“Cell%Cycle”%Test%%
The Cell Cycle assay can be used to assess the proliferation states of a monodisperse
population of cultured mammalian cells, categorizing cells into three general phases of mitosis:
G0/G1, S, and G2/M phases. It is important to note that the cell cycle assay on Moxi GO™ has
been configured to run specifically with the Orflo Technologies, LLC Cell Cycle Protocol, thereby
insuring that the sample fluorescence intensity is appropriately matched to the fluorescent gain
settings of the assay/instrument. Beyond that, it is also critical, with all cell cycle preps, to
prepare the cells into a single cell suspension. Achieving this may require protease treatments
and mechanical trituration to break apart clusters. It may also be necessary to filter the sample
to remove more stubbornly aggregated cell clusters.
To run a Cell Cycle assay, touch the associated icon from the Home screen (image below/left).

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Page 15
The user is then prompted to select a “Control” test (image above/middle). Note: As
interpretation of samples with arrested cell proliferation can be challenging, the Moxi GO™
system is designed to allow initial gating of a “Control”, or reference, sample that can be used to
establish the expected locations of the G0/G1, S, and G2/M phases. Any subsequent “Treated”
samples can be referenced to the “control” sample to use the pre-defined cycle phase gates. In
selecting a Control sample, three options are available:
a. Run a new Control test – Selecting this option will let the user run a new Control test
and establish the corresponding cell cycle phase gate ranges for that Control.
b. Select a saved test as Control – Selecting this option will present the user with the
Saved Test listing, filtered to show only previously run Cell Cycle Control tests, to
allow for the selection of a previously run Control.
c. Use prior test as Control – This selection auto-populates the last-run Cell Cycle
Control test, saving the step of having to search for the file in the Saved Test dialog.
This is particularly useful for batch analysis mode in which multiple treated samples
will be compared to a single Control file.
Following selection of the Control file, the system will make sure that a new cassette has been
properly inserted or will prompt the user to enter a new cassette. At this point, the user can also
adjust the fluorescence gain (image above/right), changing it from “default” to “low” as
necessary for brighter samples. Once the cassette is entered and start is pressed, the system
will auto-align the laser to the cassette aperture. Following alignment the user will be prompted
to open the door and enter the Cell Cycle sample into the cassette loading well. Once loaded,
the user can close the door to start the test. The initial display shows a scatter, or “dot”, plot of
the 561nm/LP, Propidium Iodide (PI) fluorescence vs. size (Coulter Principle-measured
diameter). There are two general paths at this point depending on the type of test being run:
Control Samples vs. Treated Samples. Both test workflows and outputs are described below.
Control'Sample'
Upon completion of the control sample acquisition, a Logicle-scale histogram of the PI
fluorescence is shown (image below/left). At this point, the user should touch-and-drag the
gray gate markers to tightly enclose the cell population. Note: The cell population will appear

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at the rightmost (brightest, or highest fluorescence MFI) peak(s) cluster on the histogram. After
gating the cell population, hitting the “Next” button brings the user to linear-scaled view (image
below/middle) of that gated region or region of interest (ROI). This is the view that will be used
to define the cell cycle phases. Prior to gating those phases, the user can further refine the ROI
by either using the arrows immediately below the x-axis to change the upper and/or lower scale
ranges or by hitting Back to go back to the Logicle scale view for more-coarse adjustments.
When satisfied with the scaling, the user can then touch-and-drag the green markers to enclose
the left-most peak that is the G0/G1 region. Touching Next then enables the blue gate markers
(image below/right), allowing for the demarcation of the G2/M region. Note, in gating these
regions, it is often helpful to refer to the “ratio” column in the table below (red brackets in image
below/middle and below/right). Gates should be adjusted such that the G2/M region is ~2x the
fluorescence ratio of the G0/G1 region. The calculated cell counts, percentages, MFIs, and MFI
ratios (to the G0/G1 region MFI) for each cell cycle region are presented in the table below the
histogram. Hitting Save will then save the control file and auto-prompt the user to insert a new
cassette to run a Treated sample. If no additional samples need to be run, the user can simple
touch Cancel to exit back to the Home screen.
Treated'Sample'
After running a new control sample, the user is automatically prompted (image below/left/top) to
load a new cassette to run a treated sample. Treated samples can also be run asynchronously
by selecting a prior run test (image below/left/bottom) to use as a control. Either way, upon
completion of the treated sample acquisition, a linear-scale view of the PI fluorescence
histogram is presented (image below/right). The extents of this region (x-axis scale) are
predefined by the control sample to which this sample is being compared. Likewise, the G0/G1
and G2/M gating are predetermined by the control file reference. All relevant information for
those regions is presented in table below the histogram, including: the calculated cell counts,
percentages, MFI’s, and MFI ratio’s (to the G0/G1 region MFI).