title: Studying interactions of Staphylococcus aureus with neutrophils by flow cytometry and time lapse microscopy.
abstract: We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca(2+). Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation. To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSMα operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.
authors.affiliation: Medical Microbiology, University Medical Center Utrecht, The Netherlands.
authors.forename: Bas G J
authors.initials: BG
authors.lastname: Surewaard
authors.forename: Jos A G
authors.initials: JA
authors.lastname: van Strijp
authors.forename: Reindert
authors.initials: R
authors.lastname: Nijland
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods*; D009504:Neutrophils / Q000187:drug effects* / Q000382:microbiology*; D013211:Staphylococcus aureus / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D059008:Time-Lapse Imaging / Q000379:methods*
chemical_list: D001427:Bacterial Toxins; C025971:staphylococcal delta toxin
references.citation: Lancet. 2010 May 1;375(9725):1557-68
references.pmid: 20206987
references.citation: J Immunol. 2005 Sep 15;175(6):3907-19
references.pmid: 16148137
references.citation: J Innate Immun. 2010;2(6):560-75
references.pmid: 20587998
references.citation: Mol Cell. 2008 Oct 10;32(1):150-8
references.pmid: 18851841
references.citation: Cell Host Microbe. 2010 Jun 25;7(6):463-73
references.pmid: 20542250
references.citation: Cell Microbiol. 2013 Aug;15(8):1427-37
references.pmid: 23470014
references.citation: J Vis Exp. 2012 Nov 04;(69):e4318
references.pmid: 23149597
references.citation: Clin Microbiol Rev. 2010 Jul;23(3):616-87
references.pmid: 20610826
references.citation: PLoS Pathog. 2012;8(3):e1002606
references.pmid: 22457627
references.citation: J Exp Med. 2004 Mar 1;199(5):687-95
references.pmid: 14993252
references.citation: Semin Immunopathol. 2012 Mar;34(2):237-59
references.pmid: 22080185
references.citation: J Immunol. 2006 Dec 1;177(11):8017-26
references.pmid: 17114475
references.citation: J Immunol. 2009 Nov 15;183(10):6569-78
references.pmid: 19846866
references.citation: PLoS One. 2008 Jan 09;3(1):e1409
references.pmid: 18183290