title: Single-cell and population NF-κB dynamic responses depend on lipopolysaccharide preparation.
abstract: BACKGROUND
  Lipopolysaccharide (LPS), found in the outer membrane of gram-negative bacteria, elicits a strong response from the transcription factor family Nuclear factor (NF)-κB via Toll-like receptor (TLR) 4. The cellular response to lipopolysaccharide varies depending on the source and preparation of the ligand, however. Our goal was to compare single-cell NF-κB dynamics across multiple sources and concentrations of LPS.
  
  
  RESULTS
  Using live-cell fluorescence microscopy, we determined the NF-κB activation dynamics of hundreds of single cells expressing a p65-dsRed fusion protein. We used computational image analysis to measure the nuclear localization of the fusion protein in the cells over time. The concentration range spanned up to nine orders of magnitude for three E. coli LPS preparations. We find that the LPS preparations induce markedly different responses, even accounting for potency differences. We also find that the ability of soluble TNF receptor to affect NF-κB dynamics varies strikingly across the three preparations.
  
  
  CONCLUSIONS
  Our work strongly suggests that the cellular response to LPS is highly sensitive to the source and preparation of the ligand. We therefore caution that conclusions drawn from experiments using one preparation may not be applicable to LPS in general.
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California, United States of America.
authors.forename: Miriam V
authors.initials: MV
authors.lastname: Gutschow
authors.forename: Jacob J
authors.initials: JJ
authors.lastname: Hughey
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Ruggero
authors.forename: Bryce T
authors.initials: BT
authors.lastname: Bajar
authors.forename: Sean D
authors.initials: SD
authors.lastname: Valle
authors.forename: Markus W
authors.initials: MW
authors.lastname: Covert
mesh_terms: D016475:3T3 Cells; D000818:Animals; D004926:Escherichia coli / Q000378:metabolism; D008024:Ligands; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D015398:Signal Transduction; D059010:Single-Cell Analysis; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
chemical_list: D008024:Ligands; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D018124:Receptors, Tumor Necrosis Factor; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4