Removal of endotoxin from culture media by a polymyxin B sepharose column. The activity of contaminating endotoxin in culture media measured by the interleukin 1 inducing effect on human monocyte cultures and by the Limulus test.
The in vitro study of monocytes (Mo) poses several problems. Minor contamination with endotoxin (ET) of media and utensils as well as adherence to glass or plastic surfaces may activate the cells and cause pronounced production of monokines. Many commercially liquid culture media were found to contain ET in concentrations above 25 X 10(-12) g/ml. A simple system for the removal of ET from media and solutions was established by use of a commercially available Polymyxin B Sepharose gel. To measure the lipopolysaccharide (LPS) binding capacity of the gel, known concentrations of LPS were added to culture media, which were passed through a column consisting of the Polymyxin B Sepharose gel. The content of ET and added LPS in media was measured by the Limulus amoebocyte lysate (LAL) test before and after passage of the column. The LPS-binding capacity of the gel was approximately 2.4 X 10(-6) g/10 ml. The biological activity of contaminating ET and added LPS in media, before and after passage of the column, was also characterized by the capacity of the media to induce interleukin 1 (IL-1) secretion in human Mo cultures. The content of IL-1 in Mo culture supernatants was determined by the mouse thymocyte costimulatory (LAF) assay. By comparison of the activity of ET in these different biological systems, it was demonstrated that 15-20 X 10(-12) g/ml of ET stimulate human Mo cultures to IL-1 secretion.
Mølvig
J
J
Steno Memorial Hospital, Gentofte, Denmark.
Baek
L
L
D000328:Adult; D002846:Chromatography, Affinity / Q000379:methods*; D003470:Culture Media / Q000032:analysis*; D004731:Endotoxins / Q000302:isolation & purification*; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000302:isolation & purification; D011112:Polymyxin B; D011113:Polymyxins; D012685:Sepharose
D003470:Culture Media; D004731:Endotoxins; D007375:Interleukin-1; D008070:Lipopolysaccharides; D011113:Polymyxins; D012685:Sepharose; D011112:Polymyxin B