2
Requirements for Injection Port Loading
1. To ensure proper injection in all wells during your assay,
each series of ports must contain the same injection
volume (for example, all Port A injection ports must be
filled with the same injection volume across the entire XF
Sensor Cartridge). This applies to Background Correction
wells also.
2. All compounds/reagents should be diluted with the ap-
propriate assay media before being loaded into the sensor
cartridge, unless noted otherwise. For further details,
consult the appropriate Agilent Seahorse XF Assay Kit/
Reagent user manual.
NOTE:
Serum or BSA containing solutions should not be
loaded into the ports.
3. The hydrated XF Sensor Cartridge must remain in the XF
Utility Plate, placed flat on the work surface throughout the
loading procedure. Do not lift or angle the XF Utility Plate/
XF Sensor Cartridge away from the work surface while
loading.
4. Handle the XFe96 sensor cartridge carefully. Hold the base
of the XF Utility Plate when transporting an XF Sensor Car-
tridge. To mitigate the accidental leaking of injection solu-
tions prior to starting the assay, load the injection ports in
close proximity to the Seahorse XFe96/96 Analyzer.
5. A manual or automatic pipet may be used. Ensure any
pipet is properly calibrated to accurately dispense volumes
of 20.0 to 30.0 µL.
6. The port loading guide, provided with each XF Sensor
Cartridge, can ensure the desired port is loaded. Use of
the port loading guide is encouraged, but not required. The
top of the XF Sensor Cartridge can be easily marked with
a laboratory pen to provide a visual guide. Avoid marking
any barcodes or labels on the XF Utility Plate or XF Sensor
Cartridge.
Figure 2
XFe96 Sensor Cartridge orientation.
Seahorse
A
C
E
G
B
D
F
H
Loading the XFe96 Sensor Cartridge with
injection solutions
NOTE:
The hydrated XF Sensor Cartridge must remain in the
XF Utility Plate, flat on the work surface throughout the load-
ing procedure. Do not lift or angle the XF Utility Plate/XF Sen-
sor Cartridge away from the work surface while loading. Hold
the base of the XF Utility Plate when handling the XF Sensor
Cartridge to avoid triggering any leaking of solutions from the
injection ports.
Step 1
Prepare and warm injection solutions to 37°C.
NOTE:
Injected solutions should be at pH 7.3 - 7.4 at 37°C
prior to loading into the injection ports. Diluting compounds
in Agilent Seahorse XF DMEM, pH 7.4 or XF RPMI, pH 7.4 will
facilitate accurate pH of injection solutions.
Step 2
Orient the XFe96 Sensor Cartridge as shown in Figure 2.
Place row labels (lettered A-H) to the left. The triangular notch
(green arrow) will be in the bottom left-hand corner
(Figure 2).
