Endotoxins, also known as lipopolysaccharides (LPS), are part of the outer membrane of the cell wall of Gram-negative bacteria.
Endotoxin is invariably associated with Gram-negative bacteria, regardless of whether the organisms are pathogenic. Although the term is occasionally used to refer to any cell-associated bacterial toxin, in bacteriology it is properly reserved to refer to this lipopolysaccharide complex. Gram-negative bacteria containing these endotoxins include Escherichia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus influenzae, Bordetella pertussis, and Vibrio cholerae (Figure 1).
—Kenneth Todar, at the University of Wisconsin-Madison Department of Bacteriology, On-line textbook of Bacteriology
Figure 1. Endotoxins can influence cell growth, cell differentiation, contractility and protein expression. They trigger the release of tumor necrosis factor (TNF), interleukins-1,6 and 8 and the production of platelet-activating factors, Prostaglandin E and Thromboxane A.1,2
Endotoxins Can Be Measured Using Two Methods
- Measuring a clotting reaction between the endotoxin and a clottable protein in the amoebocytes of Limulus polyphemus, the horseshoe crab
- A much more sensitive photometric test based on a Limulus amoebocyte lysate (LAL) and a synthetic color-producing substrate. The LAL assay is used for the routine check of LPS in biological solutions and was the first detection method to be certified by the FDA. LPS contamination is usually expressed in endotoxin units (EU). Typically, 1 mg LPS corresponds to 1–10 EU.
All endotoxin testing for the Purelink® HiPure Kits was performed by an independent FDA-qualified third party testing facility.
Cytotoxicity measures cell death in the presence of transfection reagents and is dependent on cell type, transfection reagent used, and plasmid DNA purity. Plasmid DNA purified with PureLink® HiPure Plasmid Kits causes very low cytotoxicity (Figure 2).
Figure 2. Low cytotoxicity of plasmid DNA purified with the PureLink® HiPure Plasmid Kits. CHO-K1 cells were grown in 96-well plates to 80% confluence, then transfected with 200 ng/well of pEF4-luciferase reporter plasmid, using 0.1 μg increments (up to 1.0 μg) of Lipofectamine™ 2000 Transfection Reagent. At 24 hr post-transfection, the cells were treated with 10 μM camptothecin for 1 hr, then assayed using the fluorescence-based Vybrant® Cytotoxicity Assay Kit. Results for an untreated control sample (“Cells”) and cells transfected with plasmid DNA only (no transfection reagent added) are also shown. PL: plasmid DNA purified using the PureLink® HiPure Plasmid Filter Maxiprep Kit. Q: plasmid purified using the Qiagen EndoFree® Plasmid Maxi Kit.
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