Seahorse XF96 User manual

XF96 Extracellular Flux Analyzer
and Prep Station
Installation and Operation Manual
100900-400_MANUAL XF96 W/ PREP STATION_RA

100900-400_MANUAL XF96 W/ PREP STATION_RA 2
First Edition:
February 2009, SHB & GWR
Revision history:
REV_A RELEASED PER ECO #0161 02/11/10 JD
Copyright:
Copyright ©2009 Seahorse Bioscience Inc. All rights reserved.
Trademark Information:
Microsoft, Windows, Excel, and Vista are registered trademarks of Microsoft
Corporation. All other trade names are properties of their respective companies.
Patents applied for:
Seahorse Bioscience Inc.
16 Esquire Road
N. Billerica, MA 01862
[phone]
[phone] Fax
Northwest Research Center
16000 Mill Creek Blvd., Suite 205
Mill Creek, WA 98012
425-338-4703
Submit technical support questions or comments about our documentation to:
[email protected]
For the latest information on products, visit our web site at:
www.seahorsebio.com
Printed in U.S.A.
General Assay Flow Chart for XF96 Assays

100900-400_MANUAL XF96 W/ PREP STATION_RA 3

100900-400_MANUAL XF96 W/ PREP STATION_RA 4
Quick Reference
XF96 Analyzer Protocol
**Please refer to the XF96 Manual for detailed instructions**
1. Pre-incubate sensor cartridges overnight in XF96 Calibrant solution in a non-CO2
incubator at 37ºC.
2. Leave XF96 Analyzer on overnight with XF96 software running and logged in to
ensure temperature is at 37ºC on the day of the assay.
3. Warm all media and compounds to 37ºC and adjust pH to 7.4. Filter to ensure
sterility.
4. Using the Prep Station, perform XF Assay media change on cell plate and incubate in
a non-CO2incubator at 37ºC for ~20 minutes before running on XF96 Analyzer. Final
volume per well should be 150-200 µl.
5. Load XF Assay template into XF96 software or make a new template using the Assay
Wizard. Use the table below to program the optimal mix, wait and measure cycle times.
When working with a new or unknown cell line, start with a 2 minute mix and 5 minute
measure cycle. Verify instrument protocol commands, export preferences and any
instrument settings that may have changed.
6. Load 25 µl pre-warmed compounds (diluted in XF Assay media) at pH 7.4 into
appropriate ports on the sensor cartridge. Optimal injection volume is 25 µl. All wells of
each port used need to be loaded with either compound or media control, including
temperature correction wells. If the sensor cartridge is allowed to cool down (more than
5 minutes), return to incubator for 10 minutes to allow it to heat back up before starting
the calibration.
7. Load sensor cartridge and Utility plate into the right side of the instrument tray.
8. After calibration is complete, replace Utility plate with the pre-incubated cell plate.

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Table of Contents
Quick Reference XF96 Assay Protocol 3-4
Table of Contents 5
Introduction 9
1. Seahorse Bioscience XF96 Instrument Overview 9
2. Technical Specifications 10
3. XF Instrument, Controller and PS Installation Procedure 12
Unpacking and Component identification 12
Suitable Locations for the instrument 16
Removing the Shipping Cartridge 17
Internal Components of the XF96 18
Set-up and Interconnects: Cable Installation 19
Keyspan Adapter Installation and Configuration 20
Unpacking the Prep Station 22
Installing the PS Manifold and Connecting the Hose Systems 22
Testing the Prep Station 27
Temperature settings for the PS incubator 28
Safety Considerations 29
4. Basic Operation of the XF96 Instrument 30
Power and Warm-up 30
XF Software and Login 31
Introduction to the XF software and Graphic User Interface 32
Cleaning and routine maintenance 38
Performing the XF Assay 39
5. Seahorse Bioscience Cell Culture Overview 39
6. XF assay Day 1 - Seeding Cells in XF96 Cell Culture Plates 39
7. XF assay Day 1 - Preparing Sensor Cartridge for the XF96 Assay 40
8. XF assay Day 2 - Preparing Cell Plate for the XF96 Assay 41
Media Changes and Maintenance of cells growing in XF plates 41