In Vitro Bioassay of Endotoxin Using Fluorescein as a pH Indicator in a Macrophage Cell Culture System.
10.3349/ymj.2005.46.2.268
- Author:
Dong Hee LEE
1
;
Hak Joon SUNG
;
Dong Wook HAN
;
Min Sub LEE
;
Gyu Ha RYU
;
Maki AIHARA
;
Kosuke TAKATORI
;
Jong Chul PARK
Author Information
1. Department of Medical Engineering, Yonsei University College of Medicine, Seoul, Korea. parkjc@yumc.yonsei.ac.kr
- Publication Type:Original Article ; Comparative Study ; Evaluation Studies ; Research Support, Non-U.S. Gov't
- Keywords:
lipopolysaccharide;
macrophage;
fluorescein;
Limulus Amebocyte Lysate test;
Staphylococcus aureus;
pH
- MeSH:
Animals;
Biological Assay/*methods/standards;
Cells, Cultured;
Comparative Study;
*Contrast Media;
Culture Media/chemistry;
Endotoxins/*analysis;
*Fluorescein;
Hydrogen-Ion Concentration;
Limulus Test;
Lipopolysaccharides/analysis;
Macrophages/*chemistry;
Mice;
Research Support, Non-U.S. Gov't
- From:Yonsei Medical Journal
2005;46(2):268-274
- CountryRepublic of Korea
- Language:English
-
Abstract:
Based on the biological activity of endotoxin, we propose a possible new method for detecting endotoxin using a pH- indication system of macrophage culture media. After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction). The advantages of this LPS- detection method were compared with the Limulus Amebocyte Lysate (LAL) test by using purified bacterial LPS (Salmonella minnessota, Escherichia coli, and Pseudomonas aeruginosa). Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system. The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.
