Agilent Technologies Seahorse XFe96 User manual

Basic Procedure
A key feature of the Agilent Seahorse XFe96/XF96 Analyzer is the ability to inject
solutions during the assay and observe changes in respiration or glycolytic rates in
real time. This is accomplished by pipetting solutions into the Injection Ports on the
XF Sensor Cartridge prior to beginning an XF assay. This procedure describes the
injection port loading process which is performed the day of your assay following
overnight XF Sensor Cartridge hydration.
Recommended injection volume is 20-30 µL for XFe96/XF96 Analyzers.
Recommended Injection Solution Volumes for 10X dilution upon injection, starting
with a microplate well volume of 180 µL assay medium (Figure 1):
1. Port A: 20 µl
2. Port B: 22 µl
3. Port C: 25 µl
4. Port D: 28 µl
The composition and number of ports utilized will depend on your assay design.
Loading the Agilent Seahorse XFe96
Sensor Cartridge Injection Ports
User Guide
Seahorse
A
C
E
G
B
D
F
H
A B
C D
Port designation
for each well
Figure 1. Recommended injection volumes and port designation for each well.

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).

3
Step 3
Place the A/D port loading guide (blue lettering) flat on top
of the sensor cartridge. Orient the port loading guide so the
letter ‘A’ (Figure 3) is in the upper left-hand corner. Use your
fingertips to hold the outside edges of the port loading guide
to stabilize during loading and to prevent pipette tips from
dislodging the port loading guide (Figure 5).
Figure 5 Dispensing Injection Solutions using the port loading guide.
Figure 6. Injection Port Mechanism and Proper Port Loading Techniques- (a) Cross section of a single XF injection port. Note the open orifice at the bottom of
the port. Injection solutions are retained in the ports via surface tension. (b) Pressurized air is used to force the solution from the port during an injection. (c) Do
NOT insert pipettes tips fully into the ports if not using a loading guide. (d) If using the XF Port Loading Guide, place the pipette tips vertically into the Port Loading
Guide holes as shown. (e) If not using the XF Port Loading Guide, then allow the pipette tips to gently touch the inside of the ports, resting the side of the pipette
tips on the port rims. (f, g) Acceptable positions of injection solution in port after loading. (h) Unacceptable position of injection solution in port after loading.
Figure 4 XFe96 Sensor Cartridge Port designations.
If you are not using a port loading guide, port A is the upper-
left port in each well quadrant (Figure 4).
Step 4
Using a multi-channel pipette, first ensure tips are securely
fitted onto the pipette before pipetting injection solutions.
Position the pipette tips (filled with your injection solutions)
into the desired column in the port loading guide, and orient
the tips at a very slight angle less than 5° (Figures 5 and 6d).
Insert the tips as far as they will go without resistance into the
holes and dispense the injection solutions. Do not force the
tips completely into the holes of the port loading guide
(Figure 6c).
Seahorse
A
C
E
G
B
D
F
H
A B
C D
Port designation
for each well
e f g ha
Injection
Port
Without
Loading
Guide
5º
OR
d
With
Loading
Guide
Injection
Manifold
Injection
Port
Orifice
b c
Figure 3 Orienting the A/D port loading guide.
1
A
A
C
E
G
B
D
F
H
3 5 7 9 112 4 6 8 10 12
1
A
D
C
E
G
B
D
F
H
3 5 7 9 112 4 6 8 10 12
When not using a port loading guide, position the pipette tips
as shown in Figure 6e. Do not fully insert the pipet tips into
the port as this may result in accidentally forcing injection
solutions through the port orifice (Figure 6c).

www.agilent.com/chem/discoverxf
For Research Use Only.
Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019
Printed in the USA, April 9, 2019
5994-0239EN r2
Step 5
After dispensing the injection solutions into the ports, with-
draw the tips from the ports carefully, holding the port loading
guide throughout the procedure. Remove the A/D port loading
guide and set aside for loading port D (if needed). Do not
tap any portion of the XF Sensor Cartridge in an attempt to
remove air bubbles from an injection port. This can cause
unintended leaking of injection solutions from the injection
ports.
NOTE: Forcefully dispensing injection solutions can over-
whelm surface tension at the injection port opening and result
in leaking of the solution from the injection port.
Step 6
Switch to the B/C port loading guide (red lettering). Orient
with the letter ‘B’ (green arrow, Figure 7) in the upper left-hand
corner, and place on top of the XF Sensor Cartridge. Repeat
loading procedure outlined in steps 3-5 for ‘B’, and injection
ports. If your experimental design requires an injection from
Port D, remove the B/C port loading guide and place the A/D
port loading guide on the XF Sensor Cartridge. Repeat loading
procedure outlined in steps 3-5 for injection port D.. Remove
and discard loading guide(s) once the entire XF sensor car-
tridge has been loaded.
Step 7
Visually inspect the injection ports for even loading (Figure
8). The solution should be in the port; make sure there are
no residual drops on top of the XF Sensor Cartridge. Once all
injection solutions have been loaded according to your ex-
perimental design, carefully transfer the XF Sensor Cartridge
(together with the XF Utility Plate) to the Seahorse XFe96/
XF96 Analyzer to start your assay.
IMPORTANT: Remove all port loading guides and lids for the
XF Sensor Cartridge and XF Utility Plate before placement
into the Analyzer.
Figure 7 Orienting the B/C port loading guide.
1
A
B
C
E
G
B
D
F
H
3 5 7 9 112 4 6 8 10 12
1
A
C
C
E
G
B
D
F
H
3 5 7 9 112 4 6 8 10 12
Figure 8 Visual verification of injection port loading.