Seahorse XFp User manual

XFp Extracellular Flux Analyzer
User Manual
Part #102745-400 Rev A

First Edition:
102745-400 Rev A December 2014
Revision history:
First release December 2014
Copyright:
Copyright ©2014 Seahorse Bioscience Inc. All rights reserved.
Seahorse Bioscience Inc.
16 Esquire Road
N. Billerica, MA 01862
Submit technical support questions or comments about our documentation to:
[e-mail]
Phone support:
Global / United States
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United Kingdom
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[phone], Select Option 3
[phone], Select Option 3
0800 096 7632
0800 180 6678
[phone]
For the latest information on products, visit our web site at:
www.seahorsebio.com
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Table of Contents
1. XFp Analyzer Overview ............................................................................................................... 4
2. Technical Specifications .............................................................................................................. 5
3. XFp Analyzer Installation
Unpacking and Component Identification ................................................................................... 6
Suitable Locations for the XFp Analyzer ....................................................................................... 7
Safety Considerations ................................................................................................................... 7
4. Basic Operation of the XFp Analyzer
Power and Warm-Up .................................................................................................................... 8
Welcome Screen on First Time Power Up .................................................................................... 8
Communications and Network Connection Setup ....................................................................... 8
Turning the XFp Analyzer Off ....................................................................................................... 8
5. Navigating the XFp Analyzer software
Setting up an XFp Assay................................................................................................................ 9
Running an XFp Assay ................................................................................................................. 11
Modifying XFp Analyzer Settings ................................................................................................ 14
XFp Analyzer Diagnostics ............................................................................................................ 16
Online Help and Support ............................................................................................................ 18
Analyzing XFp Data Using Wave for Desktop ............................................................................ 19
Appendices
Appendix I: Networking Guide ................................................................................................... 20
Appendix II: Troubleshooting ..................................................................................................... 24
Appendix III: Support Contact and Ordering Information .......................................................... 25
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1. XFp Analyzer Overview
The Seahorse Bioscience XFp Analyzer measures the rate of change of dissolved oxygen and pH in the
media immediately surrounding living cells cultured in a miniplate. Changes in the extracellular media
are caused by the consumption or production of analytes by the cells. Therefore, a sensitive
measurement of the media flux can be used to determine rates of cellular metabolism with precision
and in a completely non-invasive, label-free manner.
A unique feature of XF technology is its ability to make accurate and repeatable measurements in as
little as five minutes. This is accomplished by isolating an extremely small volume (approximately 2 μL)
of media above the cell monolayer. Cellular metabolism causes rapid, easily measured changes to the
microenvironment in this small volume.
Typically, a measurement cycle is performed for 6 minutes. The media is gently mixed and the analyte
levels are then measured until the oxygen concentration drops approximately 20-30% and media pH
declines approximately 0.1-0.2 pH units. The measurement is performed using optical fluorescent
biosensors embedded in a disposable cartridge that is placed into the specially designed Seahorse XFp
Cell Culture miniplate.
Baseline metabolic rates are typically measured 3-4 times, and are reported in pmol/min for OCR
(Oxygen Consumption Rate) and in mpH/min for ECAR (Extracellular Acidification Rate). Compound is
then added to the media and mixed, and then the post-treatment OCR and ECAR measurements are
made and repeated. As cells shift metabolic pathways, the relationship between OCR and ECAR
changes.
The XFp Analyzer system includes a bench top analyzer, disposable sensor/compound delivery
cartridges, tissue-culture treated miniplates for seeding cells for analysis, calibration solution, and
Wave software.
Consumable cartridges, miniplates and calibration reagents are packaged and sold in sets under the
trade name “FluxPaks”.
Assay kits and reagents for performing gold standard XF assays are available. The XFp Cell Mito Stress
Test and XFp Glycolysis Stress Test kits contain pre-measured, verified compounds for assessing the two
major energy pathways in cells and are recommended for characterizing the metabolic phenotype of
cells using the XFp Analyzer.
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2. Technical Specifications
Model XFp
Dimensions: Width x height x depth
12” x 17” x 23”
30 cm x 43 cm x 58 cm
Weight: 33 lbs / 15 Kg
Power Requirements 100-240V AC 50/60Hz
6A/3A
Power cord rating 3- wire (grounded) AC power cord rated 10A or greater
Power fuse ratings 250V/5A Time Lag (2 fuses) 5mm X 20mm
Environmental
Conditions
‘Normal’ environmental conditions
- Indoor use, altitude to 2000 m
Room Temperature
Range
+59°F - 86°F / +15°C - 30°C
No direct sunlight
Humidity 20 - 70 % RH, non-condensing
Data Interface TCP/IP (external)
USB Type A (one in front, two in back)
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3. XFp Analyzer Installation
Unpacking and Component Identification
Upon receipt immediately check each box for damage. Report any shipping damage to the
transportation company, and Seahorse Bioscience using the contact information on page 2
of this document.
The following items are included in an XFp system:
Instrument/Controller Quantity
XFp Analyzer 1
Power Cord (region-specific) 1
Wireless Micro USB Adapter 1
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Suitable Locations for the XFp Analyzer
The XFp Analyzer is designed for laboratory use. The internal environment for the XFp Cell Culture
Miniplate is controlled to a preset temperature; therefore, laboratory room temperature must be
maintained within the range listed in the specification table in Section 2. The miniplate tray
temperature can be monitored using the status display on the upper right portion of the user interface.
The XFp Analyzer uses optical detection technology to measure extremely low levels of fluorescent
emission from analyte sensors. Therefore, although the instrument has been designed to shield room
light, excessive light (such as direct sunlight) should be avoided.
Safety Considerations
WARNING: The protection provided by this instrument may be compromised if it is used in a manner not
specified by Seahorse Bioscience.
Safe operation of the XFp Analyzer requires that all covers be securely attached and plate tray door is
closed. This also prevents heat loss and system cooling, which can affect data quality.
The door opens automatically when the tray is extended, allowing the operator to insert or remove the
assay consumables. The operator must exercise caution during the loading of the miniplate/cartridge in
order to avoid the possibility of a pinch hazard. After the miniplate/cartridge is placed securely on the
tray, the operator’s hand must be removed from the area of the tray before continuing the assay. After
the command is given to continue the assay via the user interface, the tray will move slowly back into
the instrument and the door will close. An optical sensor is used to determine the status of the door.
The XFp Analyzer has heaters around the miniplate that maintain a stable temperature. Typically, the
temperature will be maintained at 37°C, as monitored by temperature sensors and controllers
embedded in above the tray. A thermal fuse will disable the heater should it reach an abnormally high
temperature.
The user shall not replace the power cord provided with any other power cord that is rated at less than
what is specified in Section 2, dependent on the power mains of the country the instrument will be used
in.
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4. Basic Operation of the XFp Analyzer
Power and Warm Up
To power on the XFp Analyzer, toggle the
power switch on the back of the instrument.
Allow at least 20 minutes for the instrument to
fully warm and equilibrate to the set
temperature.
The status icon in the upper right corner
of the screen will display the temperature and a green check mark.
Welcome Screen on First Time Power Up
On initial power up, a welcome screen will display some initial diagnostic tests on the instrument.
Follow the on-screen directions to run them.
These tests can be run at any time by using the Diagnostics menu selection on the instrument user
interface (For more details, refer to Section 5: Navigating the XFp Analyzer Software).
XFp Analyzer Communications and Network Connection Setup
The transfer of template and result files between the XFp Analyzer and an analysis computer running
Wave can be accomplished through any of the USB connectors on the instrument. Seahorse
recommends connecting the XFp Analyzer to a local network to ease file movement and aid Seahorse
Technical Support in servicing the instrument. Please contact Seahorse Technical Support with any
questions.
Refer to the XFp Networking Guide (Appendix I) for instructions on setting up a wired network
connection as well as a wireless network connection using the included wireless adapter. NOTE: Only
the included wireless adapter has been qualified to work with the XFp Analyzer.
Turning the XFp Analyzer Off
To turn off the instrument, press the power button in the lower-left corner of the Home screen to first
shut down the XFp Analyzer screen. After the screen turns black, use the power switch on the back of
the instrument to completely shut down power.
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5. Navigating the XFp Analyzer
Setting up an XFp Assay
To set up an assay on the XFp Analyzer, select Start from the Home
screen.
1. Select Template from Local, USB or Network drive
Seahorse Bioscience provides default templates for XF Stress Test
and cell characterization assays. Templates can also be created in
Wave running on a desktop or laptop computer then transferred to
the XFp Analyzer via network connection or USB flash drive.
a. Click Start
b. Select a template provided by Seahorse Bioscience to
perform a specific assay. These templates can be found in the Local tab.
c. Templates designed in Wave can be transferred to the XFp
Analyzer via USB or Network location.
i. If the instrument is networked, a Network tab will
appear and allow selection and running of a
template from the networked location.
ii. If a USB thumb drive containing a valid XFp
template has been inserted into the instrument, a
USB tab will appear. Templates can be run directly
from this location.
Refer to the Wave User Guide for XFp (available
from the Seahorse Bioscience website) for more
information about creating and transferring
templates.
Note! Only XFp assay template files (*.asyt) are
recognized by the XFp Analyzer. Templates must reside
on the root of the USB flash drive and NOT in a subfolder.
2. Verify Groups and Plate Map
After selecting the template file, make any necessary
modifications to the Plate Map and Groups to be analyzed.
a. To see the conditions defined for a Group, touch the group
name and look at the header information, as shown.
b. To change the wells assigned to each group, touch the
Group name followed by the well(s) to be included.
c. For other changes to Groups, Wave running on a desktop
computer must be used.
Please refer to the Wave User Guide for XFp for more
detailed information about modifying Group Conditions.
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3. Review/Edit Instrument Protocol
Check that all desired steps are selected (check boxes checked).
Seahorse Bioscience strongly recommends performing
equilibration for all cell-based XF assays. If needed, increase or
decrease the number of measurements that will be performed
during the assay on the Instrument Protocol page by touching
the circled number corresponding to the step to be modified,
then adjusting the number of cycles up or down. (Refer to
Define the Instrument Protocol section of the Wave User Guide
for XFp Analyzer for more information about measurement
cycles).
4. Review Summary and Start Assay
a. Review the General Information, and Errors and Warnings
sections to verify that the settings are as desired.
b. Before beginning an assay, the following optional steps may
be taken:
i. Click Edit next to Assay Name to customize the
name of the Assay Result file.
ii. Click Edit next to Notes to add any notes related to
the assay or protocol being performed.
iii. Click Edit next to Email Notification to add email
addresses for recipients to be notified when the
assay is complete.
c. Click Start Assay then follow the on-screen instructions.
NOTE: The XFp Analyzer will save assay results to a USB flash drive, if available. Seahorse
Bioscience recommends inserting a USB flash drive into the instrument at the time of assay
setup so that results will automatically be saved.
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Running an XFp Assay
1. Load the Cartridge and Utility Plate onto the
tray when prompted. Ensure the cartridge fits
properly on the Utility Plate, the lid is removed
from the cartridge, and the direction of the
cartridge matches the image on the screen.
2. The XFp Analyzer will perform Calibration of the sensor cartridge, which takes approximately 20
minutes.
Note: Although the sensor calibration may appear to be complete after 15 minutes, the
instrument will be busy for a few more minutes. Wait for the user prompt to appear.
3. Following calibration, the XFp Analyzer tray will open and present the utility plate. Remove the
Utility Plate and load the Cell Plate. Ensure the lid is removed from the Cell Plate prior to loading
onto the XFp Analyzer tray.
4. Once the Cell Plate is loaded, touch Continue to begin the equilibration step. Viewing the
progress bar in the upper-left area of the screen can check progress of equilibration.
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5. During the assay, two views are automatically available to visualize the data being acquired by
the XFp Analyzer. Select either tab to toggle between the views.
a. Overview
The overview tab displays both OCR and ECAR as a
function of time.
Red vertical lines indicate the injections and are
labeled by injection port letter.
i. In this view the charts can be zoomed and
scaled by choosing one of the tools below the
charts:
1. Zoom
2. Move
3. Reset
ii. By default, Rate data is displayed in Group
mode. By touching
the Options button,
different data can be
displayed:
1. Show StdDev turns on display of error bars for the each Group.
2. Level Data shows the concentration data from which the rate data is
derived.
3. Well Mode shows the data from the individual wells instead of the
average of the wells in each group.
b. OCR vs. ECAR
The OCR vs. ECAR tab displays OCR on the Y-axis and
ECAR on the X-axis. Choose a single time point at
which to examine OCR vs. ECAR by using the
Measurement selector below the chart. Data are
displayed in Group Mode with standard deviations.
The scale of this chart is fixed to allow easy
comparison of values across measurements.
Note: In both the Overview and OCR vs. ECAR views, the display of
individual wells can be turned off by touching the well graphic on the
right. Background correction is always performed while the
experiment is running.
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6. Once the assay has finished, the
XFp Analyzer will prompt the user
to eject the cartridge and remove
the cell plate.
7. After the assay is complete, the data
will automatically be saved to a USB
flash drive (if inserted), the local
drive, or the networked drive (if
configured). A message will appear
indicating the location of the save
Assay Result File (*.asyr).
Note: Canceling a run during an assay may cause the XFp Analyzer to stall. If this occurs, restart
the hardware by using the power switch on the back of the instrument. After shutting down, use
the same power switch to turn the instrument ON.
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Modifying XFp Analyzer Settings
From the Start page, touch the Settings button to access the XFp
Analyzer Settings page containing:
•System Preferences
oEnvironmental Settings
oBackup Assay Results
oAuto Assay Naming
oTemplate Management
•Device Settings
oSetup Assistant
1. Environmental Settings
a. Adjust Tolerance: Increase or decrease the temperature
tolerance for the XFp Analyzer.
b. Temperature Alarm: Enable or disable an alarm to notify
users if the temperature of the XFp Analyzer falls outside
the acceptable tolerance set above.
c. Atmospheric Pressure: Customize the atmospheric pressure
set on the XFp Analyzer to
match that of the lab in which the instrument is located.
2. Backup Assay Results
a. Backup Assay Results saves a copy of each assay performed
within the previous 60 days on the local storage within the
XFp Analyzer.
i. Insert a USB flash drive. It must be inserted and ready
to use before proceeding to step 2.
ii. To export these files either individually or collectively to a
network location or USB, click the individual check box or
click Select All and then click Export.
iii. To delete, select either individual or multiple files and
click Delete.
3. Auto Assay Naming
a. Auto Assay Naming allows one to specify the default naming
convention to be followed for each Assay Result File (*.asyr)
that is created on the XFp Analyzer.
b. The order of each variable can be adjusted by dragging and
dropping the selection in the order desired. An example of the
name will be displayed above.
c. The Custom field can be used to add a keyword, instrument
name, etc. to all assay results files.
d. Click Apply to complete the process of setting up a custom
name
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4. Template Management
a. Template Management allows transfer of assay template files between the XFp Analyzer
and a Windows computer running Wave Desktop.
1) Check the box next to an individual assay template or click Select All to select all the
assay template files.
2) Click Import (from either the USB or Network tab) to import files onto the XFp Analyzer.
-OR-
b. Click Export (from XFp Analyzer Local storage) to export files to a USB or network location.
5. Device Settings
The Setup Assistant is a series of screens that guide configuration
of network settings.
Note: Seahorse recommends that the Device Settings be configured by
the institution’s IT department and reference the XFp Networking Guide
(Appendix I) for assistance.
a. Wired Network – Ethernet cable is required to set up
wired network access. The Ethernet cable port is located
on the lower backside of the XFp Analyzer
b. Wireless Network – A wireless USB adapter can be
plugged into one of the available USB ports
(recommended on lower back side of XFp Analyzer) to
gain wireless access. Once finished, select an available
SSID from the list and click Connect. If there are no visible
networks available, click Refresh.
Note: The XFp Analyzer must be restarted after the Wireless
USB adapter is plugged in.
c. Network Directory – Once network access setup has been
completed, the Network Directory allows for setting a
network location to save Assay Template and Assay Result
files. It is essential to verify the spelling of the Shared
Directory, Domain, User Name and Password for this to
function.
d. Email Configuration – Set up an email address for
completed assay notifications. Time Zone – Set the time
zone for the XFp Analyzer. Once set, the correct time will
be visible on the screen as well as when using the time
stamp function for auto assay naming.
e. Date & Time – Adjust the date and/or time. This can be
done automatically by connecting to a time server (screen
5), or set manually (screen 6).
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XFp Analyzer Diagnostics
The XFp Diagnostics page contains a variety of functions that assist
Seahorse Technical Support in debugging any issues or errors
encountered during normal operation. The System Check diagnostic
function should be performed upon initial startup of the XFp Analyzer
and upon Seahorse Technical Support request.
To access the Diagnostics page, go to the Home pages then touch
Diagnostics.
System Files – Press Send to send the system log files if the
XFp Analyzer encounters an error or issue. Seahorse Technical
Support will also request log files as necessary. The XFp
Analyzer must be networked in order to automatically send
system files to Seahorse Technical Support.
1. Diagnostic Tests
System Check - Overall System
Communication Test – Verifies all system modules are
communicating appropriately.
Voltage Test – Ensures voltage levels are within specified
ranges for operation.
Motor Test – Verifies the movements and positions of the
tray and probes
Holding Pressure Test – Ensures the injection system
does not have any leaks.
Injection Zero Test – Ensures function of the zero
pressure sensor.
Injection Solenoid Test – Verifies proper open/close operation of internal valves.
Measurement System:
LED Reference Test – Verifies system records LED intensity signal when ON.
Auto-zero Test – Analyze and record any offset values found within the channels on each
board.
Noise Test – Analyze and record the amplitude of the noise level on each channel.
High-Gain Rise Time Test – Conducts an amplifier rise time test.
Thermal Performance:
Thermal Test – Verifies that the temperature remains within tolerance after a “Wait”
period.
Advanced Measurement:
Drift Test – Tests stability of each channel.
LED Drift Test – Tests stability of each LED.
LED Noise Test – Analyze the amplitude of the noise level of each LED when ON.
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2. Consumable Check
Barcode Test – Requires user interaction to complete test. Test verifies XFp Analyzer can
read barcodes.
Calibration Test – Performs Calibration procedure and will verify all wells are calibrated
correctly.
Injection Test – Verifies proper performance of Injection
step.
3. Remote Assistance – Seahorse Technical Support has the ability to
remotely access each XFp Analyzer that has an active network
connection.
1. To perform a remote session with Seahorse Support first call
the appropriate geographically located support line then click
Start.
2. Next, an ID and Password will be presented; Seahorse
Technical Support will request this information to connect to
the XFp Analyzer.
4. Maintenance - The XFp Analyzer is designed to require minimal
cleaning and maintenance. All consumables are disposable and
none of the instrument components are exposed to the cell plate
at any time, preventing cross-contamination of biological or
chemical materials. However, the user, if necessary can perform
some tasks.
Probe Cleaner – Unavailable until June 2015.
Tray Movement – Press Tray Out to open the door and
eject the tray. Press Tray In to return the tray inside the
XFp Analyzer.
Probe Movement – Probe Movement will adjust the
probes within the XFp Analyzer up or down.
Cartridge Eject – Use this function to Eject or Load a
cartridge into the XFp Analyzer*.
*Note: If a cartridge is suspected to be inside the XFp Analyzer, use this function to remove the cartridge
before beginning another assay.
Air filter replacement: At approximately one-year intervals, the air filter at the upper rear of the
analyzer should be replaced. The filter retainer is held in place by magnets and can be pulled away from
the housing by hand. [Replacement filters can be ordered using part number: 102799-000]
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Online Help & Support
To access the Help page, go to the Start page then
touch Help.
The following information is contained on the Help screen:
1. Support
Worldwide Seahorse Support contact
information.
2. Version of Software/Hardware
Currently installed software and firmware
versions on XFp Analyzer. If the XFp Analyzer is
networked, Software Updates (Product Update
Available) or Firmware Updates (Firmware
Update Available) will be clickable.
3. “Help” Text Info - Most screens on the XFp Analyzer
have a small Help button that will display Help text
related to the current screen.
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Analyzing XFp Data using Wave for Desktop
While assay design can either be done on the XFp Analyzer or in Wave for Desktop, Seahorse
recommends Assay Result files (*.asyr) be analyzed using Wave for Desktop.
Note that Wave for Desktop can be downloaded and installed on any personal computer via the
Seahorse Bioscience website.
http://www.seahorsebio.com/support/software/update.php
Refer to the Wave User Guide for XFp (Seahorse Document 103244-400) for a detailed description of
how XFp data can be analyzed.
Additional information and protocols for preparing medium associated with XF assays, experimental
design, running XF assays and analyzing XF data may be found online at
http://www.seahorsebio.com/resources/documentation/basicprocedures/index.php, or by contacting
Seahorse Bioscience Support [[e-mail]; 1-800-671-0633 or
001-978-671-1600 (option 3)]
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APPENDIX I – Networking Guide
Required Materials: Approved Wireless USB Adapter or Wired Connection (Ethernet cable); Network
connection settings from IT Department.
Note: XFp Analyzers are shipped with Microsoft® Security Essentials (MSE) configured for Real Time
protection and weekly scheduled virus sans using default actions for its four alert levels. This will provide
sufficient protection from common threats originating in network connections and removable media;
however, MSE® will not scan media automatically on connection/insertion.
1. XFp System Information
•The XFp Analyzer ships with an integrated 100Mbps Ethernet network adapter.
•The XFp Analyzer ships with a Netgear® USB Wi-Fi Adapter (only 2 models are
approved WNS 1000M and WNA 3100-100ENS)
•There is no internal Wi-Fi adapter in the instrument.
•The Ethernet (RJ-45) jack is located at base of the instrument, in the back.
•The XFp Analyzer can be connected to any Microsoft Windows®-compatible
network and the Local Area connections can be configured, as required by the network.
2. Hardware Setup
The two options for network connection are (1) wired via Ethernet cable or (2) wireless via
Wireless USB Adapter.
Wired Connection
1. Plug the wired network connection into the back of the XFp Analyzer. Ensure that the
connection is firmly seated.
2. Power the XFp Analyzer OFF
3. Power the XFp Analyzer ON
4. Wait for the temperature to display before proceeding with the Wired Setup instructions
(Section 3)
Wireless Connection
1. Plug the USB Wireless adapter into one of the available USB ports on the back of the
instrument.
2. Wait 1-2 minutes after installing the adapter and ensure that the lights on the adapter
are on or blinking.
3. Power the XFp Analyzer OFF
4. Power the XFp Analyzer ON
5. Wait for the temperature to display in the upper right corner of the home screen before
proceeding with the Wireless Setup instructions (Section 3).
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