SHORT COMMUNICATION
A Simple Method for Human Peripheral Blood Monocyte Isolation
Vol. 95(2): 221-223, Mar./Apr. 2000
Marcos C de Almeida/*, Alan C Silva, Aldina Barral, Manoel Barral Netto/+
Centro de Pesquisas Gonçalo Moniz-Fiocruz, Rua Valdemar Falcão 121, 40295-001 Salvador, BA, Brasil *Departamento de Patologia, FCS, Universidade de Brasília, DF, Brasil
We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes.
Key words: monocytes - percoll - gradient-isolation
Macrophages and monocytes are largely used in immunological research especially for the study of intracellular parasites. The ideal method for monocyte isolation combining simplicity, cheapness, purity and high yield does not exist (Seljelid & Pertoft 1981, Bennett & Breit 1994). We propose here a simple two step procedure for obtaining highly purified human monocytes.
The most common procedure is monocyte isolation by adherence after Ficoll-Hypaque purification of peripheral blood mononuclear cells (PBMC) (Bennett & Breit 1994). Monocyte isolation by adherence, although simple, has several disadvantages: high lymphocyte contamination, low flexibility, high manipulation and monocyte transient activation (Haskill et al. 1988, Bennett & Breit 1994). Lymphocyte contamination in the first hour after adherence may be high, being as high as 40-50% after two washes and 30% even after five washings. It has been reported that in the first 24 h, after four vigorous washes, 25% of remaining cells are lymphocytes (Bennett et al. 1992). The degree of lymphocyte contamination when separating monocytes by the adherence method is probably related to percentage of lymphocytes in PBMC, the amount of PBMC laid for adherence, number of washes, strength of washing and time of adhesion. These aspects can make this method variable from donor to donor and from researcher to researcher. Alternative methods are immune-selection, centrifugal elutriation and density gradients. Immune selection is too expensive for daily routine and for large volumes of blood. Centrifugal elutriation, although the method of choice for larger volumes of blood, requires expensive equipment and a specialized technician. Several kinds of density gradients are available, both continuous and discontinuous. Pumps and ultracentrifuges, in general expensive equipment, are necessary for performing continuos gradients.
We report here on the results using a two step procedure with single gradients in each step. First using a Ficoll-Hypaque gradient (density = 1.070 g/ml) and afterwards a slight hyperosmolar Percoll gradient (density = 1.064 g/ml). Percoll solutions were done as follows: first an isosmotic Percoll was prepared as usually mixing one volume NaCl 1.5 M with nine volumes of Percoll (Pharmacia, density = 1.130 g/ml). The Percoll gradient was done mixing 1:1 (v/v) isosmotic Percoll with PBS/Citrate (NaH2PO4 1.49 mM; Na2HPO4 9.15 mM; NaCl 139.97 mM; C6H5Na3O7 .2H2O 13mM; pH 7.2). Both gradients were centrifuged at 25-35°C, 400 g for 35 min. Percentage of monocytes after the Percoll gradient was higher than 90% using morphology, histochemistry or FACS analysis (Table I, Figure). The cells were viable and functional and able to be cultivated in suspension or attached to plastic, plastic bound fibronectin, collagen or laminin (not shown). Further indications of functionality were secretion of large amounts of TNF-alpha after LPS stimulation, phagocytose of latex particles and Leishmania promastigotes both in suspension and after adherence (Table II). Eighty to 90% from the monocytes laid on the Percoll gradient were recovered afterwards (Table III). The procedure yields similar results with small and large amounts of blood. When working with small amounts of blood (< 50 ml) for saving time we have used leukocyte rich plasma after dextran sedimentation.(Meerschaert & Furie 1994). When working with very large amounts of blood (> 200 ml) was better to take the buffy coat. It has been previously proposed a two step Percoll gradient for monocyte isolation with 90% of purity but with variable yield (Seljelid & Pertoft 1981). The most important pitfall of their method was that they advocate the use of defribrinated blood. This could lead to serious cell loss and activation. As they had observed, monocytes bind strongly to small microscopic blood clots. Platelets could also bind to monocytes forming clumps (Weyrich et al. 1996). The adequate blood anticoagulation is then critical. The use of sodium citrate in all the solutions until the Percoll gradient avoids the use of defibrinatrion and platelet binding to monocytes (Roos & de Boer 1986), possibly the use of EDTA could have the same effect although we have not tested it. Platelet elimination can be easily done with low speed centrifugation (100 g) before or after the Percoll gradient although it implies in cell loss. Temperature is also a critical point. We prefer working during all the procedure at room temperature (25-35°C) as it has been shown that monocyte tends spontaneously to aggregate at lower temperatures (Mentzer et al. 1986) and platelets to be activated (White & Krivit 1967, Oliver et al. 1999). Finally as it has been shown (Fluks 1981, Boyum 1983) the monocyte purity can be improved by hyperosmotic density gradients. In conclusion the procedure devised here can be done with usual reagents and equipment of average laboratory, it is easily handled and provides a 90% pure population of monocytes.
ACKNOWLEDGEMENTS
To Silvia A Cardoso and Jorge C Andrade for technical assistance.
Fig | Table I | Table II | Table III
This work was supported by Pronex and TMRC (AI-30639, NIH-USA). MCA was supported by a Capes fellowship. MBN and AB are Senior Investigators of CNPq.
+Corresponding author. Fax: +55-21-356.2593. E-mail: mbarral@cpqgm.fiocruz.br
Received 24 June 1999
Accepted 13 December 1999
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