Lack of cations in flow cytometry buffers affect fluorescence signals by reducing membrane stability and viability of Escherichia coli strains Journal Article


Author(s): Tomasek, Kathrin; Bergmiller, Tobias; Guet, Cǎlin C
Article Title: Lack of cations in flow cytometry buffers affect fluorescence signals by reducing membrane stability and viability of Escherichia coli strains
Affiliation IST Austria
Abstract: Buffers are essential for diluting bacterial cultures for flow cytometry analysis in order to study bacterial physiology and gene expression parameters based on fluorescence signals. Using a variety of constitutively expressed fluorescent proteins in Escherichia coli K-12 strain MG1655, we found strong artifactual changes in fluorescence levels after dilution into the commonly used flow cytometry buffer phosphate-buffered saline (PBS) and two other buffer solutions, Tris-HCl and M9 salts. These changes appeared very rapidly after dilution, and were linked to increased membrane permeability and loss in cell viability. We observed buffer-related effects in several different E. coli strains, K-12, C and W, but not E. coli B, which can be partially explained by differences in lipopolysaccharide (LPS) and outer membrane composition. Supplementing the buffers with divalent cations responsible for outer membrane stability, Mg2+ and Ca2+, preserved fluorescence signals, membrane integrity and viability of E. coli. Thus, stabilizing the bacterial outer membrane is essential for precise and unbiased measurements of fluorescence parameters using flow cytometry.
Journal Title: Journal of Biotechnology
Volume: 268C
Publisher: Elsevier  
Date Published: 2018-02-20
Start Page: 40
End Page: 52
DOI: 10.1016/j.jbiotec.2018.01.008
Notes: We thank R Chait and M Lagator for sharing Bacillus subtilis CR_Y1 and pZS*_2R-cIPtet-Venus-Prm, respectively. We are grateful to T Pilizota and all members of the Guet lab for critically reading the manuscript. We also thank the Bioimaging facility at IST Austria for assistance using the FACSAria III system.
Open access: no