SciELO - Scientific Electronic Library Online

 
vol.95 issue2Role of CD8+ T cells in endogenous interleukin-10 secretion associated with visceral leishmaniasisPolymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1 author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276On-line version ISSN 1678-8060

Mem. Inst. Oswaldo Cruz vol.95 n.2 Rio de Janeiro Mar./Apr. 2000

http://dx.doi.org/10.1590/S0074-02762000000200014 

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

linha2.jpg (2100 bytes)

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. \

 

 bul1.gif (879 bytes) ACKNOWLEDGEMENTS

To Silvia A Cardoso and Jorge C Andrade for technical assistance.

 

 bul1.gif (879 bytes) REFERENCES

  • Bennett S, Breit SN 1994. Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV. J Leukoc Biol 56: 236-240.
  •         [ Links ]
  • Bennett S, Por SB, Stanley ER, Breit SN 1992. Monocyte proliferation in a cytokine-free, serum-free system. J Immunol Methods 153: 201-212.
  •         [ Links ]
  • Boyum A 1983. Isolation of human blood monocytes with Nycodenz, a new non-ionic iodinated gradient medium. Scand J Immunol 17: 429-436.
  •         [ Links ]
  • Fluks AJ 1981. Three-step isolation of human blood monocytes using discontinuous density gradients of Percoll. J Immunol Methods 41: 225-233.
  •         [ Links ]
  • Haskill S, Johnson C, Eierman D, Becker S, Warren K 1988. Adherence induces selective mRNA expression of monocyte mediators and proto-oncogenes. J Immunol 140: 1690-1694.
  •         [ Links ]
  • Meerschaert J, Furie MB 1994. Monocytes use either CD11/CD18 or VLA-4 to migrate across human endothelium in vitro. J Immunol 152: 1915-1926.
  •         [ Links ]
  • Mentzer SJ, Guyre PM, Burakoff SJ, Faller DV 1986. Spontaneous aggregation as a mechanism for human monocyte purification. Cell Immunol 101: 312-319.
  •         [ Links ]
  • Oliver AE, Tablin F, Walker NJ, Crowe JH 1999. The internal calcium concentration of human platelets increases during chilling. Biochim Biophys Acta 1416: 349-360.
  •         [ Links ]
  • Roos D, de Boer M 1986. Purification and cryopreser-vation of phagocytes from human blood. Methods Enzymol 132: 225-243.
  •         [ Links ]
  • Seljelid R, Pertoft H 1981. In DO Adams, PJ Edelson & HS Koren (eds), Methods for Studying Mononuclear Phagocytes, Academic Press, p. 201-205.
  •         [ Links ]
  • Weyrich AS, Elstad MR, McEver RP, McIntyre TM, Moore KL, Morrissey JH, Prescott SM, Zimmerman GA 1996. Activated platelets signal chemokine synthesis by human monocytes. J Clin Invest 97: 1525-1534.
  •         [ Links ]
  • White JG, Krivit W 1967. An ultrastructural basis for the shape changes induced in platelets by chilling. Blood 30: 625-635.
  •         [ Links ]

 

Fig | Table I | Table II | Table III

linha2.jpg (2100 bytes)

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

Creative Commons License All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License