Resources - Application Notes

Normalization of Agilent Seahorse XF Data by In-situ Cell Counting Using a BioTek Cytation 5


Related Products: Cytation 5

May 16, 2017

Authors: Yoonseok Kam, Ned Jastromb, Brian P. Dranka, Agilent Technologies, Inc.; Joe Clayton, Paul Held, BioTek Instruments, Inc.

Agilent Technologies



The Agilent Seahorse XF analyzer is a platform that measures cellular metabolic activity in multiwell microplates. Data normalization between different experimental conditions is commonly achieved using either protein assay or cell counting. We evaluated an XF data normalization protocol combining the in situ nuclear staining capability of the XF analyzer with BioTek Instrument’s Cytation 5 system, which is able to count cellular objects in situ. Once the XF analysis is completed, an injection of membrane-permeable Hoechst 33342 from one of the remaining injection ports of the XF cartridge stains nuclei in situ. The fluorescently labeled nuclei can be imaged and counted on the Cytation 5 with no additional sample processing. In our trials using three morphologically distinct cell lines, we performed normalization of the basal oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). The outcomes were highly comparable to protein assay. The workflow combining the post-XF analysis in situ nuclear staining and intact cell counting by Cytation 5 is a faster, more reliable data normalization procedure, and is also highly expandable for further downstream analysis.