Isolation of rat peritoneal mononuclear and polymorphonuclear leucocytes on discontinuous gradients of Nycodenz.
Centrifugation of rat leucocytes from thioglycollate-induced inflammatory peritoneal exudate on a discontinuous gradient of Nycodenz with a density of 1106 g/l and an osmolarity of 400 mosmol/l separated the polymorphonuclear from the mononuclear leucocytes. The cells on the interphase between the buffer and the gradient medium contained 96% mononuclear leucocytes with a recovery of greater than 60%, and the bottom fraction consisted of 98% polymorphonuclear leucocytes with a yield of 91%, when an exudate isolated 20 h after the injection of thioglycollate was fractionated. Leucocytes isolated from non-inflamed rat peritoneum could be enriched in the fraction of nonspecific esterase-positive cells from 86% to 96% with a recovery of 82% on a gradient with a density of 1091 g/l and an osmolarity of 325 mosmol/l. The viability of the isolated cells was greater than 95% (trypan blue exclusion test), and there was no measurable reduction in the fraction of phagocytosing cells (latex and opsonized zymosan) after exposure to the hypertonic gradient material.
Institute of Physiology, University of Aarhus, Denmark.
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D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002499:Centrifugation, Density Gradient / Q000379:methods*; D004950:Esterases / Q000378:metabolism; D007472:Iohexol; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000201:enzymology; D008297:Male; D009504:Neutrophils / Q000187:drug effects; D010529:Peritoneal Cavity / Q000166:cytology*; D010587:Phagocytosis; D051381:Rats; D011919:Rats, Inbred Strains; D013864:Thioglycolates / Q000494:pharmacology; D015054:Zymosan / Q000494:pharmacology
D013864:Thioglycolates; D007472:Iohexol; D015054:Zymosan; D004950:Esterases