In vitro bioassay of endotoxin using fluorescein as a pH indicator in a macrophage cell culture system.
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.
Lee
Dong Hee
DH
Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea.
Sung
Hak-Joon
HJ
Han
Dong-Wook
DW
Lee
Min-Sub
MS
Ryu
Gyu Ha
GH
Aihara
Maki
M
Takatori
Kosuke
K
Park
Jong-Chul
JC
D000818:Animals; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D002478:Cells, Cultured; D003287:Contrast Media; D003470:Culture Media / Q000737:chemistry; D004731:Endotoxins / Q000032:analysis*; D019793:Fluorescein; D006863:Hydrogen-Ion Concentration; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000032:analysis; D008264:Macrophages / Q000737:chemistry*; D051379:Mice
D003287:Contrast Media; D003470:Culture Media; D004731:Endotoxins; D008070:Lipopolysaccharides; D019793:Fluorescein