GeminiBio Orflo Moxi GO II User manual

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

This document is provided to customers who have purchased Orflo Technologies, LLC (“Orflo”, a subsidiary of
Gemini BioProducts, LLC) 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 II™ is intended for research use only. NOT for use in diagnostic procedures. 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
shall not, nor shall it permit any third party to: (i) use, copy, modify, translate or adapt the Software or Documentation
other than as expressly permitted by the Terms of Sale, which can be found here
(http://www.orflo.com/v/vspfiles/pages/resources.html); (ii) rent, lease, share, disclose or transfer the Software,
provide it on a subscription basis, or use it in connection with a service bureau or other configuration whereby any
third party may use the Software, (iii) create any derivative works of or based on the Software or Documentation; (iv)
reverse-engineer, decompile, disassemble or otherwise attempt to discern the source code of the Software; (v)
remove, relocate, alter or obscure any trademark, copyright or other proprietary or restrictive marking or legend on
the Software or Documentation or any copies thereof; or (vi) use, provide or disclose the Software in violation of any
applicable laws, orders or regulations.
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
owners. Any such usage is intended only to designate those manufacturer’s products as supplied by Orflo for
incorporation into its equipment and does not imply any right or license to use or permit others to use such
manufacturer’ and or their product names as trademarks.
© 2020 Gemini BioProducts, LLC All rights reserved.
Rev. 2.10, 02/2020
Orflo Technologies and Moxi GO II™ are trademarks of Gemini BioProducts, LLC, registered in the U.S. and other
countries.
Windows, Windows XP, Windows Vista, Windows 7, and Windows 8, Windows 10 are trademarks of Microsoft
Corporation, registered in the U.S. and other countries.
Mac and Mac OS are trademarks of Apple Corporation, registered in the U.S. and other countries.
FlowJo is a trademark of Treestar, Inc.
Flowmi™ is a trademark of Bel-Art.
Bluetooth® and the Bluetooth logos are registered trademarks of Bluetooth SIG, Inc., registered in the U.S. and other
countries.
Accutase® and Accumax® are registered trademarks 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 GO II™ SYSTEM OVERVIEW ......................................................................................................................... 4
MOXI GO IITM ACCESSORIES .................................................................................................................................. 5
GETTING STARTED ................................................................................................................................................ 6
GENERAL GUIDELINES .......................................................................................................................................... 6
ADDITIONAL MATERIALS REQUIRED ..................................................................................................................... 7
USING THE MOXI GO IITM ...................................................................................................................................... 8
HOME (START) SCREEN .................................................................................................................................................. 8
SETTINGS ................................................................................................................................................................... 10
Date and Time .................................................................................................................................................... 10
Passcode Lock ..................................................................................................................................................... 10
Fluorescence Gain .............................................................................................................................................. 11
Auto Noise-Find .................................................................................................................................................. 11
Auto Gate settings .............................................................................................................................................. 11
SAMPLE PREPARATION CONSIDERATIONS ......................................................................................................................... 11
RUNNING A TEST ......................................................................................................................................................... 13
General Instructions (All Tests except “Cell QC”, “Open Flow” and “Multiplex Bead Assay”) ............................ 13
• Inserting a Cassette: ................................................................................................................................................ 14
• System Calibration: ................................................................................................................................................. 14
• Loading a sample: .................................................................................................................................................... 15
“Open Flow Cytometry” Test .............................................................................................................................. 16
“GO Flow” Tests (Simplified version of “Open Flow”) ......................................................................................... 18
“Cell QC” Test ..................................................................................................................................................... 19
Cell QC – Single Run .......................................................................................................................................................... 19
Cell QC – Start Batch ......................................................................................................................................................... 20
Cell QC – Initial Test Output (both Single Run and Batch) ................................................................................................ 21
Cell QC – Test Output – Continuing Batch Testing ............................................................................................................ 22
Cell QC – Batch Run – Saved Data and .CSV Summary File ............................................................................................... 23
Cell QC – “Add to Existing Batch” ...................................................................................................................................... 24
Multiplex Bead Assay ......................................................................................................................................... 24
Overview – Current State .................................................................................................................................................. 24
Running a Multiplex Reference Sample ............................................................................................................................ 25
Running a Multiplex Unknown Sample ............................................................................................................................. 27
Locating all Unknown tests linked to a “Reference” sample ............................................................................................ 28
TEST RESULTS ............................................................................................................................................................. 30

Recorded Channels ............................................................................................................................................. 30
Data Display Formats – General Output Types .................................................................................................. 30
Fluorescence vs Size Scatter Plot ...................................................................................................................................... 31
Histograms ........................................................................................................................................................................ 32
Fluorescence vs. Fluorescence Scatter Plot ...................................................................................................................... 33
Reading Raw Data Output (Conc., Size, MFI, and %) ......................................................................................... 34
Fluorescence vs. size scatter (dot) plot ............................................................................................................................. 34
Single Channel Histogram Plot .......................................................................................................................................... 34
Fluorescence vs. Fluorescence Scatter (Dot) Plot ............................................................................................................. 35
Simplified Output and Auto-Gating Overview .................................................................................................................. 35
“PBMC Check" Output ........................................................................................................................................ 36
“CAR-T Expansion" Output ................................................................................................................................. 37
“Cell QC” – Output (both Single Run and Batch) ................................................................................................ 39
Quadrant – Data Summary Table Output (Apoptosis, GFP, Cell Health) ............................................................ 40
ANALYZING/GATING THE DATA ...................................................................................................................................... 40
(General) Recommended Post-Test Workflow ................................................................................................... 41
Re-scaling the X axis (size range) ....................................................................................................................... 42
Gating the data .................................................................................................................................................. 42
Overview ........................................................................................................................................................................... 42
Size Gating of Scatter (Dot) Plots ...................................................................................................................................... 44
Fluorescent Gating of Scatter (Dot) Plots ......................................................................................................................... 44
Noise Gating of Scatter (Dot) Plots ................................................................................................................................... 45
“Web” or “Quadrant” Gating – Fluorescence vs. Fluorescence Scatter Plots ................................................................... 46
Store and Recall of Gate Settings ...................................................................................................................................... 47
Histogram Overlays on Fluorescence vs Size Scatter Plots ............................................................................................... 48
“Size Histogram” Assay ..................................................................................................................................................... 48
Changing Display Axis for Tests (PMT vs Size, Histogram Only Views, “Web” or Quadrant Views) ................... 50
FILE/TEST OPERATIONS ................................................................................................................................................ 52
Test Naming and Renaming Tests ...................................................................................................................... 52
Saving and Deleting Data/Tests ......................................................................................................................... 52
Exporting Data Screenshots ............................................................................................................................... 53
Editing Saved Tests ............................................................................................................................................. 53
Compare/Overlay Saved Tests ........................................................................................................................... 55
COMPENSATION (OF SPILLOVER) .................................................................................................................................... 57
Overview/Background ........................................................................................................................................ 57
Moxi GO IITM Compensation ............................................................................................................................... 57
General Workflow ............................................................................................................................................................. 57
Standard – Single Test - Compensation ............................................................................................................................ 58
Compensation with Negative Control (Load Control Set) ................................................................................................. 61
Compensation – Future Tests ........................................................................................................................................... 62
EXPORTING DATA – CONNECTING VIA USB (USB ON-THE-GO) ............................................................................................ 64
Connect the Moxi GO IITM to a Computer ........................................................................................................... 64
Put the Moxi GO IITM in USB Connect mode ....................................................................................................... 65
Safely disconnect Moxi GO IITM from the Computer ........................................................................................... 66
MOXI GO IITM HELP AND SYSTEM INFORMATION .............................................................................................................. 66
MOXI GO IITM APPS .................................................................................................................................................... 67
INSTRUMENT FIRMWARE AND SOFTWARE UPGRADES ...................................................................................... 69

MOXI GO II – TRAY INSERT REPLACEMENT PROCEDURE ..................................................................................... 71
BACKGROUND ............................................................................................................................................................. 71
SYSTEM REQUIREMENTS AND ORDERING INFORMATION ..................................................................................................... 71
REMOVING AN EXISTING INSERT ...................................................................................................................................... 72
INSTALLING AN INSERT .................................................................................................................................................. 74
TROUBLESHOOTING ........................................................................................................................................... 76
MAINTENANCE AND STORAGE ........................................................................................................................... 80
SPECIFICATIONS FOR THE MOXI GO II™ AND CASSETTES ...................................................................................... 83
ORDERING INFORMATION ................................................................................................................................. 85
TECHNICAL AND CUSTOMER SERVICE ................................................................................................................. 85
WARRANTY ........................................................................................................................................................ 86
APPLICABLE PATENTS ......................................................................................................................................... 87

Moxi GO II™ User Guide
Page 1
Introduction
The Moxi GO II™ from Orflo Technologies (a subsidiary of Gemini BioProducts, LLC) is a
benchtop instrument that utilizes a disposable thin-film cassette and a combination of a 488 nm
laser, two photomultiplier tubes (525/45 nm filter with either a 561nm LP filter or 646nm/LP filter,
user-swappable), Coulter Principle-based cell measurements, and on-board software to provide
easy-to- run applications and data analysis. The instrument relies on disposable cassettes for
sample handling, which alleviates the need for flow cell cleaning and fluidics maintenance. The
instrument is small enough to be portable between the lab bench and the hood. A touchscreen
interface and simplified GUI allows for ease of operation without extensive training.
The Moxi GO II enables highly accurate cell counts, cell sizing, quantitative assessments of cell
viability, apoptosis, immunophenotyping, and GFP checks for a broad range of cellular analysis.
Data acquisition occurs within approximately 5-10 seconds per test. The instrument can store
up to 4Gb of data. Data is provided in the industry-standard Flow Cytometry Standard (FCS
v3.1) format as well as user-generated bitmap screenshots. Data can be downloaded to a PC
or Mac via USB connectivity.
IMPORTANT NOTE:
The Moxi GO II™ is intended for research use only. Not for use in diagnostic procedures.
It is not intended for use in medical diagnostic or therapeutic procedures, and it has not been
approved or cleared by the United States Food and Drug Administration for such uses.
About the User Guide
The Moxi GO II™ User Guide provides detailed information for operating, maintaining, and
troubleshooting the Moxi GO II, 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. Please refer the Protocols section
(http://www.orflo.com/v/vspfiles/pages/protocols.html) of the Orflo website for more details on
sample preparation.
To update the Moxi GO II firmware to the latest version, please visit http://www.orflo.com, and
register your instrument by entering a valid email address and serial number. The registration
key provided after registering will allow users access future firmware updates.

Moxi GO II™ User Guide
Page 2
Conventions Used in the User Guide
Within this manual, the following conventions are used to call out certain points, and also to
reminder users of areas of precaution for safety reasons.
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 II™ 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. IT 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.
• 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 II. Aside from swapping of
filters and replacement of cassette tray inserts, 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 II is a Class 1 laser product in accordance with IEC 60825-
1:2007. The system contains a Class IIIb laser that operates at 488 nm with

Moxi GO II™ User Guide
Page 3
a maximum output power of 60 mW. 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 II if there is any damage to the instrument’s enclosure.
• Do not attempt to disassemble or service the Moxi GO II 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 II™ User Guide
Page 4
Moxi GO II™ System Overview
The Moxi GO II™ Kit includes the Moxi GO II system, USB Cable (mini USB B to USB A 2.0),
USB Power Adapter, one pack of 25 Type S+ (MXC030) Cassettes, the extra 646nm/LP filter, a
spare cassette tray insert, and a Quick Start Reference Guide. The following figure shows the
main visible components of the Moxi GO II and the table provides a description of each
component.
Power/Reset
Button
TouchScreen
Display
Cassette Tray
Door
Cassette Tray
Latch/Lever
USB Cable Port
(on back surface)
Swappable Filters
for PMT’s
Disposable Cassette

Moxi GO II™ User Guide
Page 5
Component Function
Touch Screen Display Allows user to interface with the instrument by pressing
icons and targets. 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 ~10 seconds.
Cassette Tray Door The door is manually opened and closed by user to allow
insertion of a cassette into cassette tray. Door must be
closed prior to running a test.
Cassette Tray Latch Manually depressed by user to open cassette tray and
allow cassette insertion and removal. Release of lever,
with cassette in place, latches cassette into position for
acquisition
USB Cable Port Connects instrument to USB cable.
Swappable Optical Modules Custom optical detection filters for testing with various
dyes/fluorophores.
Cassette Disposable cassette used for loading samples. Each
cassette contains two fill ports, one at each end, thereby
allowing for two tests to be run per cassette.
Moxi GO IITM Accessories
The Moxi GO II™ uses a USB Cable and Power Adapter for power source. The Moxi GO II 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.
Power Adapter (US style) USB Cable Replacement Cassette Tray Insert

Moxi GO II™ User Guide
Page 6
Part
Function
Power Adapter, US Style
Connects USB cable to a 110 VAC outlet
USB Cable
Connects instrument to Personal computer
(PC or Mac) or power adapter
Cassette Tray Insert
Extra tray insert can be user-installed in
cassette tray when adhesive build-up
interferes with insertion/removal of cassettes.
Getting Started
The Moxi GO II™ is shipped in a condition ready for initial use; though we recommend checking
for software updates and updating the Moxi GO II with the most current software version upon
receipt. To begin, plug the USB Cable into the instrument at one end (mini-USB). 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.
CAUTION Use only an 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.
NOTES Tests should only be performed with the instrument powered by an AC
source.
The device will power on and users can access files without attaching to a
power outlet but for data collection the device must be plugged into an
electrical outlet.
General Guidelines
The Moxi GO II is used with Type S+ or MF-M Cassettes. Refer to the Specifications section for
information on the operating size and concentration ranges for the system.
The sample volume for a test should be 60 - 75 µL.
Cells should be suspended in 0.9% salt solution (e.g. PBS or equivalent) to ensure proper
conductivity for unit operation and to ensure proper particle sizing. Water, hypotonic, or
hypertonic solutions are not acceptable diluents.

Moxi GO II™ User Guide
Page 7
Cells need to be prepared as single-cell suspensions. Clusters/aggregates should be broken
apart with mechanical trituration (e.g. pipetting vigorously) and/or protease dissociation (e.g.,
Accutase® or Accumax®). For stubbornly aggregated samples, pre-filtration with a 40 µm cell
strainer is recommended.
As the Moxi GO II™ uses a 488 nm laser with 525/45 nm and either (561nm/LP or 646nm/LP,
user-swappable) detection filters, dye/fluorophore selection should be based accordingly. A few
examples for each include:
PMT Filter
Compatible Fluorophore Examples (not complete list)
525/45 nm
FITC, Alexa Fluor® 488, BB515, GFP, Calcein, Acridine Orange,
BODIPY 500/510, Rhodamine 110
561nm/LP
PE, PI, PE-Cy5, PerCP-Cy5, 7-AAD, PerCP, DsRed
646nm/LP
PI, 7-AAD, PE-Cy5
For assays other than “Open Flow Cytometry” and “GO Flow”, using ORFLO optimized and
validated protocols are strongly recommended. Please see
(http://www.orflo.com/v/vspfiles/pages/protocols.html) for a list of currently available protocols.
For cell counting application, cells can be run without fluorochromes using any of the assays.
Additional Materials Required
• Cell or bead sample (diluted and dissociated, if necessary); 60 µL minimum.
• Type S+ (MXC030, recommended for almost all assay) or MF-M (MXC010) Cassettes
• Pipette (typically P100 or P200) and appropriately sized pipette tips
• Cell staining/labeling reagents (test specific, see guidelines in “Sample Preparation”)

Moxi GO II™ User Guide
Page 8
Using the Moxi GO IITM
Press the Power Button to turn on the instrument. The Home Screen will become available.
The image below identifies the key items on the Home Screen.
Home (Start) Screen
NOTE: The home page appearance may change as additional applications are launched and
new GUIs (graphical user interface buttons) are added to the Moxi GO II software program.
Assays
/Applications
Help
Instructions
System
Settings
System
Applications
Saved
Data/Tests
Battery/
Charging
Indicator
USB
Indicator
USB Disk Mount
(for data
transfer)
Date/
Time

Moxi GO II™ User Guide
Page 9
Depending on the initial purchased configuration of the Moxi GO II system, certain
assays/applications may be disabled (examples in images below). These assay can be
enabled by contacting Orflo/Gemini ([email protected], 855-879-6694).
Assays
/Applications
Grayed out
assays are
“locked.”
Please
contact
Orflo/Gemini
to unlock.
(855-879-6694,
[e-mail])

Moxi GO II™ User Guide
Page 10
Settings
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Set the date and time by pressing the Settings icon on the main menu of the instrument. Then
touch the Date or Time field. Use the up/down arrow keys to change the values.
+",,-.'$%/.-0%
The Moxi GO II can be setup to require a password login to use the instrument and access
stored data. To set the Password lock, select the Settings icon and then touch the “Off” entry to
toggle it on (See images below). Select Done. The system will present a keyboard to enter the
four-digit numeric passcode.

Moxi GO II™ User Guide
Page 11
On entering the passcode, the user will be required to “Re-enter Passcode” to prevent
accidental entry of a wrong code.
Upon successful re-entry of the passcode, the system will display a “lock enabled” message.
The system will now require a passcode for access on initial startup and after entering sleep
mode.
NOTE: In order to turn the Passcode Lock off, the user will be required to enter the
correct passcode. If you forget this passcode, the entire system will have to be reset and
data could be lost. Keep the passcode recorded in a safe place to prevent loss of data.
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The “Fluorescence Gain” field sets the level of fluorescence gain for the “Open Flow Cytometry”
and “GO Flow” assays only. Five possible values (Very Low, Low, Default, Medium, High) can
be entered here and will seed the value of the dropdown list on the “Open Flow Cytometry” and
“GO Flow” start screens. To change the values simply tap on the current value and the system
will toggle the gains with each tap. Note: the user can also make a one-time (per-test) change
to the fluorescent gain value of the “Open Flow Cytometry” and “GO Flow” assays by touching
the value on the test start screen as described in “Running a Test” “Open Flow Cytometry Test”
section (step 6).
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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. With the
v2.10 (or later) release of the firmware, it is recommend that this setting is left in the “Off”
setting.
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The user is given control in setting the system auto-gating to one of three settings:
1. Off – the system will not auto-gate any tests.
2. Viability Assays – the system will auto-gate the Viability tests only: Cell QC, PBMC
Check, CAR T Expansion and Cell Count (Size + Viability).
3. All Assays – the system will auto-gate the first PMT vs. Size display output for all tests.
To toggle the auto-gate value, simply tap on the current setting for that field.
Sample Preparation Considerations
There are several key considerations for preparing a sample for analysis with the Moxi GO II™:

Moxi GO II™ User Guide
Page 12
1. Solution Conductivity – As the system utilizes an electrical measurement for counting
cells, sizing cells, and metering the fluid volume, cells need to be suspended in a
conductive media. The target solution conductivity would be 0.9% salt solution (e.g.
PBS or equivalent). Deviations of +/- ~50% conductivity can be tolerated by the system.
Most medias that are isotonic to PBS will work.
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:
i. Type S+ cassette: 3 - 27 μm diameter (14 – 10,306 fL volume)
ii. MF-M cassette: 4 - 35 μm diameter (14 – 22,449 fL volume)
b. Concentration range:
i. Type S+ Cassettes:
1. Counts: 1 x 104 – 1.75 x 106 total particles/ml
2. Optimum Fluorescence Sensitivity: < 5 x 105 particles/ml
ii. MF-M Cassettes:
1. Counts: 1 x 104 – 7.5 x 105 total particles/ml
2. Optimum Fluorescence Sensitivity: < 3 x 105 particles/ml
Optimal concentration ranges up may vary slightly depending upon your cell
or bead type.
3. Single Cell Suspensions – While the Type S+ and MF-M Cassettes do have a pre-filter
for removing sporadic large particles, cell samples should be prepared as single cell
suspensions to avoid clogging of cassettes. Clusters/aggregates can be broken apart
with mechanical trituration and/or protease dissociation (e.g. Accutase® or Accumax®).
Larger particles or stubbornly aggregated clusters can be removed with a 40 µm cell
strainer. Standard centrifuge tube strainers can be used for larger volumes or a pipette-
tip FlowMi™ strainer can be used for smaller volume filtering.
4. Fluorescent Labeling/Stains - The Moxi GO II™ uses a 488 nm laser for excitation
with 525/45 nm and either 561 nm/LP or 646nm/LP (second filter is user-swappable)
detection filters. Dye and fluorophore selections should be based accordingly
PMT Filter
Compatible Fluorophore Examples (not complete list)
525/45 nm
FITC, BB515®, Alexa Fluor® 488, GFP, Calcein, Acridine Orange,
BODIPY 500/510, Rhodamine 110
561nm/LP
PE, PE-Cy5, PerCP-Cy5, 7-AAD, PerCP, PI, DsRed
646nm/LP
PI, 7-AAD, PE-Cy5
5. Post-Incubation Washes for fluorescent labels – To optimize sensitivity of the
fluorescent signal, it is recommended (for most samples) that the sample be washed 2x
(minimum) with a minimum of 1.5ml staining buffer or PBS to remove the excess (free)
antibody.

Moxi GO II™ User Guide
Page 13
6. Red Blood Cell (RBC) containing samples – For samples with high levels of RBC’s
(e.g. peripheral blood), it is necessary to eliminate/reduce the level of RBC’s in order to
prevent those cells from “masking” the cells of interest. The preferred approaches to
purification of a sample are density-gradient centrifugation (e.g. Ficoll-Paque®), magnetic
bead separation, or “pre-plating” (culturing of adherent cells). RBC lysis approaches can
be applied but users should be aware that those approaches have limited efficiency and
can still leave a significant number of intact RBC’s behind.
For assays other than “Open Flow Cytometry” and “GO Flow”, using ORFLO optimized and
validated protocols are strongly recommended. Please see website
(http://www.orflo.com/v/vspfiles/pages/protocols.html) for a list of currently available protocols.
For cell counting application, cells can be run using any of the assays.
Running a Test
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• Please follow sample preparation guidelines above (“Sample Preparation
Considerations”) in preparing the sample. Note: The minimum volume required for a
test is 60 µL.
• Turn the instrument on by pressing the power button and the Home screen will be
displayed. Select a test type by pressing one of the icons displayed on the screen.
o NOTE: The instrument must be plugged in to an AC source to run a test. Tests
CAN NOT be run on internal battery power alone or computer USB power.
• Basic test instructions are presented for all tests (example for “Viability Test” shown
below/left). For two PMT tests, a compensation menu (image below/middle and
below/right) is presented to provide the option to select a previously-used (“Use Prior”)
compensation setting. For the “Open Flow Cytometry” and “GO Flow” tests, the
additional option of setting the “Fluorescence Gain” (on a per-test basis) is presented
(image below/right). Touch the gray box (red arrow in image) to change this gain. Five
options, in order of increasing gain for the fluorescence signal output, are possible: Very

Moxi GO II™ User Guide
Page 14
Low, Low, Default, Medium, and High. The initial value populated in this field is
determined by the global “Fluorescence Gain” setting that can be set from the Settings
icon off of the Home Screen.
• !"#$%&'"()*)+*##$&&$, Open the Front Door and depress the Cassette Tray Lever (image
below). Slide a test cassette, with ORFLO lettering-side up into the cassette tray (either
end 1 or 2). Release the Cassette Tray Lever and close the Door. Note: If the cassette
had been previously used, the system will generate a “Used or Invalid Cassette” error
message.
• -.#&$/)+*0'1%*&'2", The system will automatically perform a calibration step as outlined
in the images below. The automated sequence aligns the laser to the cassette flow cell
(“Aligning the laser”) and prompts the user to open the door. Notes:
a. System status messages are displayed in the black bar at the top of the
screen.
b. For the first test-run on the unit, following power up, an initial laser “cam
align” is performed (see image below/left). Following that initial test, only the
standard laser alignment (images below/middle and below/right) are
performed.
c. Should the alignment result in an error make sure that your cassette is
pushed into the sled. And, make sure the cassette is not inserted at an
angle.

Moxi GO II™ User Guide
Page 15
• 32*4'"()*)#*/50$, System messages at the top of the screen prompt the user to “Open
the Door” (image below/left) and pipette a 60 – 75 µL sample (“Enter the Sample”, image
below/middle) into the fill port of the cassette (either test 1 or test 2, depending on which
end of the cassette was inserted into the instrument) and close the Door.
Notes:
• To load the sample, place the pipette tip directly in the loading oval (touching the
cassette with the tip is perfectly fine and recommended).