GeminiBio ORFLO Moxi V User manual

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

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 2 of 108
Title: Moxi V – 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 V™ 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/documents/termsandconditions.pdf); (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.
© 2021 Gemini BioProducts, LLC All rights reserved.
Rev. 2.12, 06/2021
Orflo Technologies and Moxi V™ 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.

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 3 of 108
Title: Moxi V – User Guide
Contents
INTRODUCTION ................................................................................................................................................... 1
ABOUT THE USER GUIDE ...................................................................................................................................... 1
CONVENTIONS USED IN THE USER GUIDE ............................................................................................................ 2
SAFETY PRECAUTIONS ......................................................................................................................................... 2
ELECTRICAL SAFETY ........................................................................................................................................................ 2
LASER SAFETY ............................................................................................................................................................... 3
BIOLOGICAL SAFETY ....................................................................................................................................................... 3
MOXI V™ SYSTEM OVERVIEW .............................................................................................................................. 5
MOXI VTM ACCESSORIES ....................................................................................................................................... 7
GETTING STARTED ............................................................................................................................................... 8
GENERAL GUIDELINES .......................................................................................................................................... 8
ADDITIONAL MATERIALS REQUIRED .................................................................................................................... 9
USING THE MOXI VTM .......................................................................................................................................... 10
HOME (START) SCREEN ................................................................................................................................................. 11
SETTINGS ................................................................................................................................................................... 12
Fluorescence Gain ............................................................................................................................................... 12
Date and Time .................................................................................................................................................... 12
Auto Noise-Find .................................................................................................................................................. 13
Auto Gate settings .............................................................................................................................................. 13
Idle Shutdown Timer ........................................................................................................................................... 13
Password Expiration ........................................................................................................................................... 13
Secure Mode ....................................................................................................................................................... 13
SAMPLE PREPARATION CONSIDERATIONS ......................................................................................................................... 13
RUNNING A TEST ......................................................................................................................................................... 15
Instructions – Cell Count (Size + Viability), PBMC Check, CAR T Expansion Tests ............................................... 15
• Inserting a Cassette: ................................................................................................................................................ 15
• System Calibration: ................................................................................................................................................. 15
• Loading a sample: .................................................................................................................................................... 16
“Cell QC” Test ..................................................................................................................................................... 18
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 ............................................................................................................ 23
Cell QC – Batch Run – Saved Data and .CSV Summary File ............................................................................................... 24
Cell QC – “Add to Existing Batch” ...................................................................................................................................... 25

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 4 of 108
Title: Moxi V – User Guide
TEST RESULTS ............................................................................................................................................................. 25
Recorded Channels ............................................................................................................................................. 25
Data Display Formats – General Output Types .................................................................................................. 26
Fluorescence vs Size Scatter Plot ...................................................................................................................................... 27
Histograms ........................................................................................................................................................................ 29
Reading Raw Data Output (Conc., Size, MFI, and %) .......................................................................................... 31
Fluorescence vs. size scatter (dot) plot ............................................................................................................................. 31
Single Channel Histogram Plot .......................................................................................................................................... 32
Simplified Output and Auto-Gating Overview .................................................................................................................. 32
“PBMC Check" Output ........................................................................................................................................ 33
“CAR-T Expansion" Output ................................................................................................................................. 35
“Cell QC” – Output (both Single Run and Batch) ................................................................................................ 36
ANALYZING/GATING THE DATA ...................................................................................................................................... 38
(General) Recommended Post-Test Workflow ................................................................................................... 39
Re-scaling the X axis (size range) ........................................................................................................................ 39
Gating the data .................................................................................................................................................. 40
Overview ........................................................................................................................................................................... 40
Size Gating of Scatter (Dot) Plots ...................................................................................................................................... 42
Fluorescent Gating of Scatter (Dot) Plots ......................................................................................................................... 43
Noise Gating of Scatter (Dot) Plots ................................................................................................................................... 44
Store and Recall of Gate Settings ...................................................................................................................................... 45
Histogram Overlays on Fluorescence vs Size Scatter Plots ............................................................................................... 46
“Size Histogram” Assay ..................................................................................................................................................... 46
Changing Display Axis for Tests (Fluorescence vs Size, Histogram Only Views) ................................................. 49
FILE/TEST OPERATIONS ................................................................................................................................................ 51
Test Naming and Renaming Tests ...................................................................................................................... 51
Saving and Deleting Data/Tests ......................................................................................................................... 51
Exporting Data Screenshots ................................................................................................................................ 52
Editing Saved Tests ............................................................................................................................................. 53
Compare/Overlay Saved Tests ............................................................................................................................ 55
EXPORTING DATA – CONNECTING VIA USB (USB ON-THE-GO) ............................................................................................ 58
Connect the Moxi VTM to a Computer ................................................................................................................. 58
Put the Moxi VTM in USB Connect mode ............................................................................................................. 59
Safely disconnect Moxi VTM from the Computer ................................................................................................. 61
MOXI VTM HELP AND SYSTEM INFORMATION .................................................................................................................... 61
MOXI VTM APPS .......................................................................................................................................................... 62
21 CFR PART 11 OPERATION (“SECURE MODE”) .................................................................................................. 64
OVERVIEW ................................................................................................................................................................. 64
ACTIVATING SECURE MODE (21 CFR, PART 11 COMPLIANT MODE) ...................................................................................... 64
SECURE MODE – KEY SETTINGS ...................................................................................................................................... 66
Idle Shutdown Timer ........................................................................................................................................... 67

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 5 of 108
Title: Moxi V – User Guide
Password Expiration ........................................................................................................................................... 67
Secure Mode ....................................................................................................................................................... 67
SYSTEM LOGIN ............................................................................................................................................................ 67
USER ADMINISTRATION ................................................................................................................................................ 68
Adding a User ..................................................................................................................................................... 68
User Roles ........................................................................................................................................................... 70
Modifying Users .................................................................................................................................................. 71
Deleting Users .................................................................................................................................................... 72
RUNNING TESTS – SECURE MODE ................................................................................................................................... 73
Saving Data ........................................................................................................................................................ 73
Electronic Signature .......................................................................................................................................................... 73
File Save Formats .............................................................................................................................................................. 75
FCS data ....................................................................................................................................................................... 75
BMP File ....................................................................................................................................................................... 75
REVIEWING DATA – SECURE MODE ................................................................................................................................. 76
SYSTEM EVENT LOGGING – SECURE MODE ....................................................................................................................... 79
Log File Naming .................................................................................................................................................. 80
Logged System Events ........................................................................................................................................ 80
BACKING UP / TRANSFERRING DATA – SECURE MODE ........................................................................................................ 81
Test-Files Directory ............................................................................................................................................. 81
Users Directory ................................................................................................................................................... 81
INSTRUMENT FIRMWARE AND SOFTWARE UPGRADES ....................................................................................... 82
UPGRADE INSTRUCTIONS: .............................................................................................................................................. 82
DATA (µSD) DISK FORMAT (STRONGLY RECOMMENDED) ....................................................................................... 83
MOXI V – TRAY INSERT REPLACEMENT PROCEDURE ........................................................................................... 86
BACKGROUND ............................................................................................................................................................. 86
ORDERING INFORMATION .............................................................................................................................................. 86
REMOVING AN EXISTING INSERT ...................................................................................................................................... 86
INSTALLING AN INSERT .................................................................................................................................................. 87
TROUBLESHOOTING ........................................................................................................................................... 89
MAINTENANCE AND STORAGE ........................................................................................................................... 94
SPECIFICATIONS FOR THE MOXI V™ AND CASSETTES ........................................................................................... 97
ORDERING INFORMATION .................................................................................................................................. 99
TECHNICAL AND CUSTOMER SERVICE ................................................................................................................. 99
WARRANTY ...................................................................................................................................................... 100
APPLICABLE PATENTS ....................................................................................................................................... 101

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 1 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 1
Introduction
The Moxi V™ 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 532 nm laser, one
PIN diode (with a 561nm/LP filter), 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 V enables highly accurate cell counts, cell sizing, quantitative assessments of cell
viability. 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 V™ 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 V™ User Guide provides detailed information for operating, maintaining, and
troubleshooting the Moxi V, 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.

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 2 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 2
To update the Moxi V 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.
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 V™ 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 V. Aside from swapping of filters
and replacement of cassette tray inserts, the instrument has no user serviceable

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 3 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 3
parts. All service must be performed by ORFLO, or an approved ORFLO
vendor.
Laser Safety
WARNING The Moxi V is a Class 1 laser product in accordance with IEC 60825-1:2007.
The system contains a Class IIIb laser that operates at 532 nm with a
maximum output power of 50 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 V if there is any damage to the instrument’s enclosure.
• Do not attempt to disassemble or service the Moxi V 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.

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 4 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 4

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 5 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 5
Moxi V™ System Overview
The Moxi V™ Kit includes the Moxi V system, USB Cable (mini USB B to USB A 2.0), USB
Power Adapter, one pack of 25 Type S+ (MXC030) Cassettes, a spare cassette tray insert, and
a Quick Start Reference Guide. The following figure shows the main visible components of the
Moxi V and the table provides a description of each component.

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 6 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 6
Power/Reset
Button
TouchScreen
Display
Cassette Tray
Door
USB Cable Port
(on back surface)
Disposable Cassette

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 7 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 7
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.
USB Cable Port Connects instrument to USB cable.
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 VTM Accessories
The Moxi V™ uses a USB Cable and Power Adapter for power source. The Moxi V 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
Part
Function
Power Adapter, US Style
Connects USB cable to a 110 VAC outlet

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 8 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 8
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 V™ is shipped in a condition ready for initial use; though we recommend checking for
software updates and updating the Moxi V 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 V 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 60µL.

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 9 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 9
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.
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 40µm cell
strainer is recommended.
As the Moxi V™ uses a 532nm laser with a 561nm/LP filtered PIN diode for detection.
Dye/fluorophore selection should be based accordingly. As the system is intended as a cell
viability system, the main recommended fluophore is Propidium Iodide (PI, recommended
2µg/ml final PI concentration). Please refer to the Orflo Viability Staining Protocol for the
staining protocol for viability assessments.
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”)

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 10 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 10
Using the Moxi VTM
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.

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 11 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 11
Home (Start) Screen
Assays
/Applications
Help
Instructions
System
Settings
Battery/
Charging
Indicator
Date/
Time
USB
Indicator
System
Applications
Saved
Data/Tests
User
Administration
(secure mode)
USB Disk
(data
transfer)

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 12 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 12
NOTE: The home page appearance may change as additional applications are launched and
new GUIs (graphical user interface buttons) are added to the Moxi V software program.
Settings
Fluorescence*Gain*
The “Fluorescence Gain” field sets the level of fluorescence gain for all the assays. Five
possible values (Very Low, Low, Default, Medium, High) can be entered here and will set the
values used for all assays. The fluorescence gain is used to control the positioning of the data
within the range of the scatter plot. “Default” gain should be used for most applications. If the
data is extending to the top of the scatter plot and “compressing” or “piling” up there, then the
fluorescent gain should be lowered. Alternatively, if the fluorescence signal is compressed
towards the bottom of the plot, a higher gain setting can be used to help increase the separation
between the fluorescence positive and negative clusters.
Date*and*Time*
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.

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 13 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 13
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. With the
v2.10 (or later) release of the firmware, it is recommend that this setting is left in the “Off”
setting.
Auto*Gate*settings*
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. ON – the system will auto-gate all tests.
To toggle the auto-gate value, simply tap on the current setting for that field.
Idle*Shutdown*Timer**
Time period for system to automatically go to sleep (and log out the user if Secure Mode is On).
Tap field to toggle values. Possible values are : 5min, 15min, 30min, 45min, 60min, 2hrs.
Password*Expiration*
Time period for system to auto-expire a User’s password. Once the password expires, the
system will force the user to update it prior to logging in. Tap field to toggle values. Possible
values are 1, 3, 6, 9, and 12 months.
Secure*Mode*
This field turns on/off the 21 CFR, Part 11 functionality for the system. See the 21 CFR Part 11
section of the User Guide.
Sample Preparation Considerations
There are several key considerations for preparing a sample for analysis with the Moxi V™:
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

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 14 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 14
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. As the Moxi V™ uses a 532nm laser with a 561nm/LP filtered PIN diode for detection.
Dye/fluorophore selection should be based accordingly. As the system is intended as a
cell viability system, the main recommended fluophore is Propidium Iodide (PI,
recommended 2µg/ml final PI concentration). Please refer to the Orflo Viability Staining
Protocol for the staining protocol for viability assessments. For cell counting application,
cells can be run without fluorochromes using any of the assays.
5. 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

Document # MXV.SYS.004
Effective: 30JUL2021
Revision # 2.12
Page 15 of 108
Title: Moxi V – User Guide
Moxi V™ User Guide
Page 15
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.
Running a Test
Instructions*–*Cell*Count*(Size*+*Viability),*PBMC*Check,*CAR*T*Expansion*Tests*
• 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).
• !"#$%&'"()*)+*##$&&$, Open the Front Door all the way and slide a test cassette, with
ORFLO lettering-side up into the cassette tray (either end 1 or 2). Close the Door.
Note: If the cassette had been previously used, the system will generate a “Used or
Invalid Cassette” error message.
• -.#&$/)+*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: