Agilent Technologies Seahorse XFe96

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.

Agilent Technologies Seahorse XFe96 Specifications

General

Model	Seahorse XFe96
Instrument Type	Extracellular Flux Analyzer
Number of Wells	96
Measurement	Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR)
Temperature Control	Yes
Cell Types	Adherent and suspension cells
Power Requirements	100-240 V AC, 50/60 Hz
Sample Throughput	Up to 96 samples per run
Format	96-well plate
Injection Ports	4 injection ports per well
Temperature	37°C
Plate Type	Seahorse XF96 microplates
Software	Wave Software
Weight	68 kg (150 lbs)
Measurement Parameters	OCR, ECAR, ATP production, Glycolysis, Mitochondrial function
Assay Time	Variable, typically 1-3 hours
Detection Method	Fluorescence (pH) and oxygen-sensitive probes

Summary

Loading the Agilent Seahorse XFe96 Sensor Cartridge Injection Ports

Basic Procedure

Describes the injection port loading process, following overnight XF Sensor Cartridge hydration.

Requirements for Injection Port Loading

Loading the XFe96 Sensor Cartridge with Injection Solutions

Step 1: Prepare and Warm Injection Solutions

Prepare and warm injection solutions to 37°C, ensuring correct pH for accurate results.

Step 2: Orient the XFe96 Sensor Cartridge

Properly orient the XFe96 Sensor Cartridge on the work surface for loading.

Step 3: Place the AD Port Loading Guide

Position the A/D port loading guide correctly on the sensor cartridge for stable loading.

Step 4: Dispense Injection Solutions

Dispense injection solutions using a multi-channel pipette with correct technique.

Step 5: Withdraw Tips and Remove Loading Guide

Carefully withdraw pipette tips and remove the loading guide after dispensing.

Step 6: Load BC and D Ports

Switch and use the B/C port loading guide for subsequent port loading.

Step 7: Visual Inspection and Analyzer Transfer

Perform visual inspection of loaded ports before transferring the cartridge to the Analyzer.