Decontaminating particles exposed to bacterial endotoxin (LPS)

Lipopolysaccharide (LPS), which comes from the cell wall of gram-negative bacteria, can stimulate murine macrophage cells to produce nitric oxide (NO), cytokines, such as tumor necrosis factor-alpha, and interleukins, such as IL-6. When examining the biological effects of particles on macrophages, it is important to have no contaminating LPS associated with the particles and none with any cell culture media or supplies since even very low levels of LPS are stimulatory. The presence or absence of LPS was observed in two ways: (1) the amount of NO produced by RAW 264.7 murine macrophage cells, and (2) the Limulus amebocyte lysate (LAL) test. Treating particles with 70% ethanol at room temperature for 48 h, followed by washing the polymethylmethacrylate (PMMA) particles with endotoxin-free phosphate-buffered saline three times, decontaminated LPS and LPS-treated PMMA particles. When given LPS that had been treated with 70% ethanol for 48 h at room temperature or at 37 degrees C, cells did not produce NO above control levels. Negative LAL tests indicated the presence of extremely low levels or the complete absence of LPS in 70% ethanol-treated LPS.