Yonsei Med J.  2005 Apr;46(2):268-274. 10.3349/ymj.2005.46.2.268.

In Vitro Bioassay of Endotoxin Using Fluorescein as a pH Indicator in a Macrophage Cell Culture System

• Lee DH
• Sung HJ
• Han DW
• Lee MS
• Ryu GH
• Aihara M
• Takatori K
• Park JC

• KMID: 1734054
• DOI: http://doi.org/10.3349/ymj.2005.46.2.268

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.

Keyword

lipopolysaccharide; macrophage; fluorescein; Limulus Amebocyte Lysate test; Staphylococcus aureus; pH

MeSH Terms

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

Figure

• 

  Fig. 1 Fluorescein and many of its derivatives exhibit multiple, pH-dependent ionic equilibria. Lowering the pH of the culture media isolated from endotoxin-stimulated macrophages could dramatically reduce the fluorescence intensity of fluorescein as a pH indicator.

• 

  Fig. 2 The pH-indicative range of fluorescein was examined, in terms of both absorption and emission, by a certified pH-standardized culture media (DMEM containing 10% FBS without phenol red; pH 4.0 to 7.0). (A) The absorption of fluorescein decreased with the reduced pH, from 0.43 at pH 7.0 to 0.12 at pH 4.0. (B) The emission intensity of fluorescein decreased significantly as the pH was reduced, from 500.7 at pH 7.5 to 145.6 at pH 4.0.

• 

  Fig. 3 Dose dependent diminution of fluorescein absorption (A) and fluorescence (B) after LPS treatment [the LPS originated from three kinds of Gram-negative bacteria, S. minnesota (•), E. coli (▪), and P. aeruginosa (▴)]. To determine the absorption of fluorescein as a pH-indicator, fluorescein was added to the reaction media to a final concentration of 25 M, and the absorbance, as related to the pH of the reaction media, was detected at 490 nm by an ELISA reader. To determine the fluorescein emission, fluorescein was also added to the media to a final concentration of 10 M, and its intensity was detected by a luminescence spectrophotometer at 430 nm of excitation and 630 nm of emission.

• 

  Fig. 4 LAL test for detection of endotoxin unit (EU) in accordance with LPS concentration. A serial dilution of 3 different LPSs [S. minnesota (•), E. coli (▪), and P. aeruginosa (▴)] was assayed using the kinetic turbidimetric method.

• 

  Fig. 5 Comparison of the macrophage culture system-method and the LAL test at minute concentrations of LPS below the unit ng/ml scale [S. minnesota (•), E. coli (▪), and P. aeruginosa (▴)]. Absorption (A) and fluorescence intensity (B) of fluorescein in the reaction media and the EU values (C) of 3 different LPSs were detected and compared with each other in the order of magnitude increases for concentration from 0.04 EU to 5 EU. The asterisk indicates statistical significance at p ≤ 0.05, as compared with untreated controls. Data represent mean values (± S.D.) of triplicate samples per condition for the experimental method.

• 

  Fig. 6 Comparison of the macrophage culture system and the LAL test, in terms of sensitivity and specificity to sonicated S. aureus (Gram-positive bacteria) extracts. Absorption (•) of fluorescein in the reaction media and EU values (▪) of the lysed S. aureus extracts were detected and compared with each other in the order of magnitude increases for concentrations from 102 CFU/ml scale to 107 CFU/ml scale (n=4).

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