1: J Immunol Methods. 1981;41(2):225-33. Three-step isolation of human blood monocytes using discontinuous density gradients of Percoll. Fluks AJ. A three step method for the purification of normal human blood monocytes is described. The procedure consists of a combination of dextran sedimentation, Ficoll-Isopaque (F-I) centrifugation and isopycnic centrifugation on discontinuous gradients of Percoll. No selective loss of monocytes was observed after the first step, and after F-I centrifugation mononuclear cells (MNC) were obtained, of which 20 +/- 6% were monocytes. The MNC were further separated on hyper-osmotic and iso-osmotic discontinuous density gradients of Percoll. The best purification of monocytes occurred on hyper-osmotic density gradients and the density interface between 1.074 and 1.066 g/ml yielded 85 +/- 7% monocytes, 13 +/- 7% lymphocytes and 1 +/- 1% granulocytes. 77 +/- 16% of the monocytes obtained after F-I centrifugation, were recovered in this interface. The purified monocytes were viable and retained their capacity to mature into macrophages. The whole procedure takes about 5 h, is reproducible and can be applied to small and large volumes (500 ml) of blood. PMID: 6267137 [PubMed - indexed for MEDLINE] 2: J Immunol Methods. 1981;40(1):1-6. Isolation of human monocytes on re-orienting gradients of Percoll. Hardin JA, Downs JT. We have developed a rapid and simple method for isolating monocytes from peripheral blood. After mononuclear cells are isolated on a Ficoll-Hypaque gradient, the monocytes are purified by sedimentation for 5 min through a re-orienting gradient of Percoll. The method is easily reproducible and yields approximately half of the monocytes in staring blood, with minimal contamination by lymphocytes. Although both monocytes and lymphocytes have similar densities, separation occurs because these cells migrate at differing rates through the gradient to reach their equilibration point. The recovered monocytes are viable and functional; over 95% of them exclude trypan blue and they adhere to glass and plastic and ingest latex particles normally. By virtue of the rapidity and gentle nature of its centrifugation steps, the method exposes cells to only very limited non-physiologic conditions. For this reason and because the monocytes are maintained in suspension, opportunities for functional alterations are minimized. PMID: 6259262 [PubMed - indexed for MEDLINE] 3: Int Rev Cytol. 1974;38(0):369-403. Cell separation by gradient centrifugation. Harwood R. Publication Types: Review PMID: 4604191 [PubMed - indexed for MEDLINE] 4: Annu Rev Biophys Bioeng. 1972;1:93-130. Physical procedures for the separation of animal cells. Shortman K. Publication Types: Review PMID: 4630834 [PubMed - indexed for MEDLINE] 6: Blood. 1985 Aug;66(2):298-301. Differential interleukin-1 elaboration by density-defined human monocyte subpopulations. Elias JA, Chien P, Gustilo KM, Schreiber AD. Interleukin-1 (IL-1) is an important immunoregulatory peptide produced by monocytes and macrophages. Because mononuclear phagocytes are morphologically and functionally heterogeneous, we examined whether they differ in their ability to elaborate IL-1. We used discontinuous Percoll gradients to obtain five density-defined human blood monocyte subpopulations. Unfractionated monocytes and their subsets were compared for their ability to stimulate thymocyte proliferation. Supernatants obtained from the denser monocytes consistently contained more IL-1 activity than did supernatants from the less dense cells. This difference in IL-1 activity was the result of differences in IL-1 elaboration, not the selective production of an inhibitor of IL-1-induced thymocyte proliferation. These data demonstrate that density-defined human monocyte subpopulations differ in their capacity to elaborate IL-1. PMID: 3874661 [PubMed - indexed for MEDLINE] 7: J Immunol Methods. 1982;48(2):199-211. Separation of human peripheral blood monocytes on continuous density gradients of polyvinylpyrrolidone-coated silica gel (Percoll). Brandslund I, Rasmussen JM, Fisker D, Svehag SE. 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. PMID: 6276470 [PubMed - indexed for MEDLINE] 8: J Immunol Methods. 1980;33(3):221-9. Separation of human monocytes on density gradients of Percoll. Pertoft H, Johnsson A, Warmegard B, Seljelid R. Monocytes from human blood have been isolated by centrifugation in Percoll. A one-step procedure has been designed to isolate the cells from 7 ml of blood. when 5% of the white blood cells are assumed to be monocytes, an estimated average yield of 100% and a purity of 20% is achieved. The contaminating cells are almost exclusively lymphocytes. By a two-step procedure the monocytes can be obtained 90% pure with an approximate yield of 35%. The cells can be used for tissue culture without washing and they display the usual properties of mononuclear phagocytes in vitro. PMID: 7373059 [PubMed - indexed for MEDLINE] 9: J Immunol Methods. 1980;32(1):31-39. Analysis of the lymphocyte distribution during Isopaque-Ficoll isolation of mononuclear cells from human peripheral blood. Hokland P, Heron I. Using chromium-labelled mononuclear cells the movements of lymphocytes in Isopaque-Ficoll (I-F) gradients were analysed. Even small percentages of ox or autologous human erythrocytes mixed with the lymphocytes caused a significant loss of lymphoid cells and this loss was augmented when higher numbers of red cells were added. Such losses could be prevented using I-F gradients of higher densities, but then neutrophils and erythrocytes were found to contaminate the mononuclear suspensions recovered. Purified B and T lymphocytes were found to move identically on the gradients, but when human erythrocytes were added to the lymphocyte suspensions to be separated, a small but significant preferential loss of T cells was observed. PMID: 7351482 [PubMed - indexed for MEDLINE] 10: J Immunol Methods. 1977;18(3-4):225-34. Two-step separation of human peripheral blood monocytes on discontinuous density gradients of colloidal silica-polyvinylpyrrolidinone. Nathanson SD, Zamfirescu PL, Drew SI, Wilbur S. Normal human peripheral blood monocytes were purified by a two-step separation. The first step, the standard Ficoll--Hypaque (F--H) buoyant density centrifugation, yielded mainly mononuclear cells, of which 24 +/- 9% were monocytes. Isopycnic centrifugation on discontinuous gradients of colloidal silica polyvinylpyrrolidinone (CS-PVP) further separated these mononuclear cells. The density interface between 1.070 and 1.060 g/ml yielded 82 +/- 7% monocytes, 5 +/- 4% granulocytes and 13 +/- 8% lymphocytes. Sixty-six percent of the monocytes obtained after F--H separation were recovered in this layer. The monocytes were intact and viable and retained their ability to phagocytose and kill Candida pseudotropicalis and to spread on glass coverslips. Motility (both random and towards a chemoattractant) was retained but was quantitatively less than after F--H separation alone. The relative purity of the monocyte population allowed assessment of major histocompatibility surface antigens by serotyping. This confirmed the presence of HLA and Ia-like antigens on monocytes. PMID: 201698 [PubMed - indexed for MEDLINE] 12: Blood. 1976 Nov;48(5):731-42. A method for the recognition and separation of human blood monocytes on density gradients. Loos H, Blok-Schut B, van Doorn R, Hoksbergen R, Brutel de la Riviere A, Meerhof L. The density distribution of human mononuclear blood leukocytes was studied in order to define the optimal conditions for the separation of monocytes and lymphocytes by isopycnic centrifugation. Under standardized conditions, two populations of cells with partially overlapping, normally distributed densities were consistently found. The cells with the lowest density were recognized as monocytes, using phagocytosis and size distribution analysis as criteria. Since the density of monocytes continuously increased during the centrifugation, optimal separation of monocytes and lymphocytes could only be achieved by limiting the time of centrifugation to 10 min at 2200 g and 4 degrees C. The separation on discontinuous density gradients decreased when the load exceeded 8 X 10(6) mononuclear cells per sq cm. Analysis of the composition of the two cell populations obtained after separation on a three-layer discontinuous gradient revealed that the contamination of the monocytes with lymphocytes was due to the partial overlapping density distributions of both cell types. A small and a large scale method for isolation of monocytes from blood on discontinuous density gradients are presented. Under the described conditions, a preparation of functionally intact monocytes can be obtained which is comparable, both in yield and purity, to those obtained by methods based on surface adherence without the drawbacks of the latter methods. PMID: 788815 [PubMed - indexed for MEDLINE] 13: J Immunol Methods. 1974 Aug;5(3):249-52. Single-step separation of red blood cells. Granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque. English D, Andersen BR. PMID: 4427075 [PubMed - indexed for MEDLINE]