Use of SYTOX green dye in the flow cytometric analysis of bacterial phagocytosis.
BACKGROUND
Fluorescein isothiocyanate (FITC) is used widely to label the targets used in flow cytometric phagocytosis assays. Unfortunately, the fluorescence intensity of phagocytosed FITC-labeled targets is influenced by changes in intracellular pH level, making quantitative measurements with this fluorophore problematic. We describe the use of SYTOX green nucleic acid stain to measure phagocytosis by flow cytometry.


METHODS
Suspensions of isopropyl alcohol-permeabilized Escherichia coli DH5alpha were stained with the SYTOX green dye and then incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytosis was determined by gating the cells.


RESULTS
Results are expressed as percentage of phagocyte-associated green fluorescent cells. The validity of the method was shown by the effects of a phagocytosis inhibitor (incubation at 4 degrees C) or enhancer (gamma interferon [IFN- gamma] treatment) being accurately assessed with this assay.


CONCLUSIONS
The method described was reproducible and provides an advantageous alternative to the use of FITC to label bacteria for the flow cytometric measurement of target uptake by phagocytic cells.
Unit of Microbiology, Department of Health Sciences, Faculty of Experimental Sciences, University of Jaén, Paraje Las Lagunillas s/n, 23071 Jaén, Spain. jgaforio@ujaen.es
J J
JJ
Gaforio
M J
MJ
Serrano
E
E
Ortega
I
I
Algarra
G
G
Alvarez de Cienfuegos
D004926:Escherichia coli / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D017737:Macrophages, Peritoneal / Q000382:microbiology / Q000502:physiology*; D009930:Organic Chemicals; D010587:Phagocytosis / Q000502:physiology*; D015203:Reproducibility of Results; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C402795:SYTOX Green