XF Data Normalization by the Agilent Seahorse XF Imaging and Normalization System

Authors: Yoonseok Kam, Kellie Chadwick, Ned Jastromb, Brian P. Dranka, Agilent Technologies, USA

Abstract

Data normalization is required for any experiment to reach a valid conclusion, particularly when variation in sample quantity is involved. For real-time cellular metabolic analysis using Agilent Seahorse XF technology, the cell quantity per well is a commonly accepted reference value for data normalization. It can be assessed directly by cell counting or indirectly by measuring biomolecules such as total protein or genomic DNA. The Agilent Seahorse XF Imaging and Normalization system integrates XF technology with a Cytation 1/5 Cell Imaging Multi-Mode reader (BioTek Instruments, Inc). In this configuration, the Cytation 1/5 is controlled by the new Agilent Seahorse XF Imaging and Cell Counting software, which provides a simplified, automated, and validated solution for microscopic image-based cell counting. This turnkey solution includes 1) in-situ administration of a cell membrane-permeable nuclear staining compound (i.e. Hoechst 33342), 2) automated imaging and cell counting optimized for XF assays, and 3) synchronization of images and cell counts into XF result files in Wave software. This solution provides an efficient and reliable method of normalization with a seamless and rapid workflow for XF Analyzer users.