Maximizing Efficiency and Performance of Seahorse XF Assays with the Bravo Automated Liquid Handling Platform

Importance of the Topic

High-throughput cell metabolism assays, such as those performed on the Agilent Seahorse XF platform, play a pivotal role in drug discovery, toxicology, and basic research into cellular bioenergetics. The preparation of samples—including cell washing, reagent dilution, and sensor cartridge loading—strongly influences the precision and reproducibility of key parameters like oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Automating these steps minimizes manual variability and frees up valuable researcher time.

Study Objectives and Overview

This application note examines the implementation of the Agilent Bravo Automated Liquid Handling Platform to execute three critical sample preparation steps for Seahorse XFe96 assays: replacing growth media with assay media (cell washing), preparing injection solutions (serial and custom dilutions), and loading these reagents into the sensor cartridge. Through comparative experiments and performance metrics (e.g., CV, Zʹ-factor), the study evaluates improvements in data consistency, assay throughput, and hands-on time reduction.

Methodology and Instrumentation

Cell Lines and Culture Conditions:

• HEK293 and HepG2 cells seeded in XF96 microplates at 3×10⁴ or 1.5–2×10⁴ cells/well, respectively.
• Culture media: DMEM or EMEM with 10% FBS and GlutaMAX (HEK293) or standard supplements (HepG2).

Upgrade of Seahorse XF Assay Workflow:

• Automated cell washing by Bravo to exchange growth medium for assay medium, controlled aspiration/dispense speeds, and tip heights to protect monolayers.
• Automated preparation of injection solutions: two-fold serial dilutions of FCCP and antimycin A/rotenone; 100-fold dilutions for compound library screens.
• Automated loading of reagents into XFe96 cartridge injection ports, ensuring accurate volumes and eliminating cross-contamination.

Instrumentation Section:

• Agilent Bravo Automated Liquid Handling Platform with heated blocks and Bravo Seahorse Assay Workbench software.
• Agilent Seahorse XFe96 Analyzer for metabolic measurements.
• BioTek Cytation 1 imager for nuclear count normalization.

Main Results and Discussion

Cell Washing:

• Automated washes preserved delicate monolayers, yielding consistent basal ECAR and OCR values across wells (CV <5%) and over multiple days.

Injection Port Loading:

• Bravo loading of MST reagents achieved highly reproducible maximal respiration responses with intra- and inter-day CVs <6%.

Dose-Response Dilutions:

• Within-plate FCCP dose responses (0–2 µM, 0.2-µM steps) exhibited linear OCR increases and CVs <10%.
• Across-plate antimycin A serial dilutions (0.002–1 µM): IC₅₀ values were consistent row-to-row and day-to-day, demonstrating precision.

Compound Library Screen:

• Seventy-two compounds tested at 10 µM; acute OCR and ECAR inhibition measured post-injection.
• Zʹ-factors averaged 0.88 (OCR) and 0.77 (ECAR), indicating robust screening performance.

Benefits and Practical Applications

Automating Seahorse XF sample preparation with Bravo:

• Reduces manual pipetting errors and well-to-well variability.
• Improves data reproducibility within and across plates.
• Decreases hands-on time by 20–60 minutes per plate, enabling greater walk-away time.

These advantages support routine compound assessment, dose-response assays, and high-content metabolic screening in pharmaceutical and academic settings.

Future Trends and Potential Uses

Integration of automation with live-cell imaging platforms and AI-driven data analysis will further streamline metabolic phenotyping workflows. Extended automation protocols could include long-term drug treatment prior to Seahorse analysis, complex multiplexed dosing matrices, and real-time assay normalization. Adoption in other cell-based platforms (e.g., 3D spheroids, organoids) is also promising.

Conclusion

The combination of the Agilent Bravo liquid handler and Seahorse XFe96 Analyzer delivers a high-precision, high-throughput workflow for cell metabolism assays. Automated washing, reagent preparation, and cartridge loading yield consistent, reproducible data, reduce manual effort, and enhance overall assay efficiency—critical factors for drug discovery and metabolic research.

Reference

Winer L., Rogers G. W., Burroughs S. E., Dranka B. P., "Maximizing Efficiency and Performance of Seahorse XF Assays with the Bravo Automated Liquid Handling Platform," Agilent Technologies Application Note, June 2023.