A modified hot phenol-based protocol for high-purity Escherichia coli lipopolysaccharide extraction: Biochemical validation, stem cell cytotoxicity, and dose dependent multi-organ inflammation in a rat model

Gram-negative bacteria contain lipopolysaccharides (LPS) in their outer membrane, which induce strong inflammatory responses. Traditional LPS extraction methods often leave residual protein and nucleic acid contamination. These impurities interfere with downstream analyses and reduce reproducibility in biological studies. This study provides a modified hot phenol method combined with enzymatic treatments using proteinase K, DNase, RNase to extract pure LPS from <i>Escherichia coli</i>. Purity was confirmed by gel electrophoresis using Coomassie blue and silver nitrate staining. The biological activity of isolated LPS was tested on mesenchymal and embryonic neural stem cells, demonstrating decreased viability. In Wistar rats, LPS injection elevated serum IL-6 but not TNFα or IL-1β. Histological examinations indicated liver, kidney, brain, and colon tissue damage post-injection. Our results show that the modified hot phenol method efficiently produces high-purity, biologically active LPS suitable for both in vitro and in vivo inflammation studies, supporting research into inflammatory processes and associated diseases.