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Removal of Endotoxin from Culture Media by a Polymyxin B Sepharose Column
Author(s) -
MØLVIG J.,
BAEK L.
Publication year - 1987
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.1987.tb02296.x
Subject(s) - polymyxin b , lipopolysaccharide , sepharose , chromatography , limulus , in vitro , chemistry , microbiology and biotechnology , secretion , biology , biochemistry , immunology , antibiotics , paleontology , enzyme
The in vitro study of monocytes (Mo) poses several problems. Minor contamination with endotoxin (ET) of media ami 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 25x10 −12 g/ml. A simple system for the removal of ET from media and solutions was established by use of a comniercially available Polymyxin B Septiarose gel, To measure the lipopolysaccharide (LFS) binding capacity of the gel. known concentrations of LPS were added to culture media, which were passed through a column consisting or the Polymyxin B Sepharose gel. The content of ET and added LPS in media was measured hy the Limulus amoehocyte lysatc (LAL) test before and after passage of the column. The LPS‐binding capacity of the gel was approximately 2.4 × 10 g/l0 ml. The biological activity of contaminating ET and added LPS in media, before and alter passage of the column, was also characterized by the capacity of the media to induce interleukin l (IL‐1) secretion in human Mo cultures. The content of IL‐l in Mo culture supernalants 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 × 10 −12 g/ml of ET stimulate human Mo cultures lo IL‐1 secretion.