title: Purification of mast cells with an improved nonsynchronous flow-through coil planet centrifuge.
abstract: A method for cell purification was designed without using high-density media which may impair membrane receptors. Rat and mouse mast cells were separated with an improved nonsynchronous flow-through coil planet centrifuge. Peritoneal cells were suspended at a concentration of 2-3x10(7) cells/ml in conditioned RPMI 1640, supplemented with 50% heat-inactivated FCS and 0.32% sodium citrate. In each separation 3 ml of cell suspension were loaded into the coiled column and elutriated at 4 degrees C. Several conditions, including the centrifugal force, revolution/rotation ratio, density of separation media, flow speed, and designs of both coiled column and flow tubes, were examined and optimized for mast cell purification. Rat mast cells were separated at the purity of 99.2%, with an average yield of 40% under sterile conditions. Nearly 90% pure mouse mast cells were harvested, despite a very low population of mast cells available in murine peritoneal cells. Purified cells were morphologically intact and discharged granules by exocytosis as indicated by electron-microscopic observations. The average histamine release with antigenic specificity was 34 and 61%, in passive sensitization in vitro and in vivo, respectively. Mast cells sensitized with mouse monoclonal IgE antibody released histamine, similar to cells sensitized with homologous antibody. This newly devised method of cell separation will be useful to purify biologically intact mast cells.
authors.affiliation: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md., USA.
authors.forename: T
authors.initials: T
authors.lastname: Okada
authors.forename: D D
authors.initials: DD
authors.lastname: Metcalfe
authors.forename: Y
authors.initials: Y
authors.lastname: Ito
mesh_terms: D000818:Animals; D015550:Cell Degranulation; D002469:Cell Separation / Q000295:instrumentation*; D002499:Centrifugation, Density Gradient / Q000295:instrumentation*; D005260:Female; D006636:Histamine Release; D008297:Male; D008407:Mast Cells / Q000166:cytology* / Q000502:physiology / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D010529:Peritoneal Cavity / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley