Isolation of Human Monocyte Populations
This unit describes the isolation of monocytes from lymphocytes by adherence, gradient sedimentation on colloidal silica particles, and flow cytometry. Because the first two methods can result in cell activation (induction of gene expression or protein secretion), and the third is technically difficult, a fourth protocol is presented which describes counterflow centrifugal elutriation. This latter procedure can be used to isolate large numbers of purified, nonactivated monocytes.
Table of Contents
- Basic Protocol 1: Isolation of Monocytes by Adherence
- Basic Protocol 2: Isolation of Monocytes by Size Sedimentation on Percoll
- Basic Protocol 3: Isolation of Monocytes by Flow Sorting
- Basic Protocol 4: Isolation of Monocytes by Counterflow Centrifugal Elutriation
- Literature Cited
Basic Protocol 1: Isolation of Monocytes by Adherence
Basic Protocol 2: Isolation of Monocytes by Size Sedimentation on Percoll
Basic Protocol 3: Isolation of Monocytes by Flow Sorting
Basic Protocol 4: Isolation of Monocytes by Counterflow Centrifugal Elutriation
Figure 7.6.1 Design of counterflow centrifugal elutriation system.
Figure 7.6.2 Configuration of sample reservoir (A) during loading of cells and (B) during elutriation.
Figure 7.6.3 Blood monocyte morphology and flow cytometric analysis for purity. Insets show postive control staining for CD3, CD20, and CD69 using peripheral blood mononuclear cells, and for CD83 using monocyte‐derived dendritic cells.
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|Fuhlbrigge, R.C., Chaplin, D.D., Kiely, J.M., and Unanue, E.R. 1987. Regulation of interleukin 1 gene expression by adherence and lipopolysaccharide. J. Immunol. 138:3799‐3802.|
|Gmelig‐Meyling, F. and Waldmann, T.A. 1980. Separation of human blood monocytes and lymphocytes on a continuous percoll gradient. J. Immunol. Methods 33:1‐9.|
|Haskill, S., Johnson, C., Eierman, D., Becker, S., and Warren, K. 1988. Adherence induces selective mRNA expression of monocyte mediators and proto‐oncogenes. J. Immunol. 140:1690‐1694.|
|Heppelston, A.G. and Styles, J.A. 1967. Activity of a macrophage factor in collagen formation by silica. Nature 214:521‐522.|
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|Smythies, L.E., Sellers, M., Clements, R.H., Mosteller‐Barnum, M., Meng, G., Benjamin, W.H., Orenstein, J.M., and Smith, P.D. 2005. Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal acitivity. J. Clin. Invest. 115:66‐75.|
|Wahl, L.M., Katona, I.M., Wilder, R.L., Winter, C.C., Haraoui, B., Scher, I., and Wahl, S.M. 1984. Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE). I. Characterization of B‐lymphocyte‐, T‐lymphocyte‐, and monocyte‐enriched fractions. Cell. Immunol. 85:373‐383.|