Separation of human peripheral blood monocytes on continuous density gradients of polyvinylpyrrolidone-coated silica gel (Percoll).
A standardized, reproducible two-step method for separation of human peripheral blood monocytes on continuous Percoll gradients has been developed. The first step involves separation of mononuclear cell on Percoll of density 1.075 g/ml and the second step separation of monocytes from lymphocytes on a continuous Percoll gradient with a starting density of 1.075 g/ml for the formation of the gradient. The average yield during a 10 month period of daily routine use has been 74 +/- 17% (mean +/- 1 S.D.), and the average purity 63 +/- 10%. Ninety to 95% of the monocytes are viable after separation as judged from trypan blue exclusion and by ingestion of latex particles and sensitized sheep erythrocytes. The separation takes about 3 h and the total number of monocytes obtained from 40 ml of blood is in the range of 10-15 x 106. The procedure has been reliable with 3-4% separation failures, mainly due to bacterial or fungal growth in Percoll suspension or media. The contaminating cells are exclusively lymphocytes, predominantly T-lymphocytes (90-95%), when citrate is used as anticoagulant. Heparin can not be used as anticoagulant, as there appears to be a dose-dependent formation of thrombocyte aggregates which contaminate the monocytes, and result in poor separation.
D000936:Antigen-Antibody Complex / Q000276:immunology; D002449:Cell Aggregation; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008214:Lymphocytes; D009000:Monocytes; D009994:Osmolar Concentration; D010587:Phagocytosis; D011205:Povidone / Q000494:pharmacology*; D011309:Preservation, Biological; D011961:Receptors, Fc / Q000276:immunology; D012822:Silicon Dioxide / Q000494:pharmacology*
D000936:Antigen-Antibody Complex; D011961:Receptors, Fc; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone