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Spontaneous aggregation as a mechanism for human monocyte purification
Abstract:
A previously unreported property of human mononuclear phagocytes is the ability of these cells to spontaneously aggregate. Fresh mononuclear cells obtained after plateletpheresis were noted to spontaneously form large cellular aggregates. Dual parameter immunofluorescence analysis demonstrated that the aggregating cells were positive for the monocyte marker CD11 (complement receptor, type 3) but were negative for the lymphocyte marker CD3 (T3 antigen). In addition, less than 5% of the nonaggregating cells were CD11+, suggesting that almost all CD11+ cells aggregated. Cellular aggregates were independent of cell concentration and formed more efficiently at 4 degrees C than at either 22 or 37 degrees C. Based on these observations, a purification procedure utilizing Ficoll-Hypaque separation, spontaneous aggregation at 4 degrees C, and transient plastic adherence resulted in a sevenfold enrichment of the CD11+ peripheral blood monocytes. Purified monocytes were contaminated with less than 2% CD3 cells. The size, growth, and adherence characteristics as well as cytologic stains indicated that the monocytes were not significantly altered by the purification procedure. Thus, spontaneous aggregation is an efficient and convenient method for the isolation of large numbers of purified monocytes.
Insights
Human mononuclear phagocytes, specifically CD11+ monocytes, exhibit spontaneous aggregation. This property enables an efficient purification method for isolating monocytes from peripheral blood.
Area of Science:
- Immunology
- Cell Biology
- Hematology
Background:
- Human mononuclear phagocytes possess previously unreported spontaneous aggregation capabilities.
- Freshly obtained mononuclear cells from plateletpheresis spontaneously form large cellular aggregates.
Purpose of the Study:
- To investigate the spontaneous aggregation property of human mononuclear phagocytes.
- To develop an efficient method for purifying peripheral blood monocytes using this aggregation phenomenon.
Main Methods:
- Dual parameter immunofluorescence analysis to identify aggregating cells using monocyte (CD11) and lymphocyte (CD3) markers.
- Optimization of aggregation conditions, noting efficiency at 4 degrees C.
- Development of a purification protocol involving Ficoll-Hypaque separation, cold aggregation, and plastic adherence.
Main Results:
- Aggregating cells were predominantly CD11+ (monocyte marker), with minimal non-aggregating CD11+ cells observed.
- The purification procedure yielded a sevenfold enrichment of CD11+ peripheral blood monocytes.
- Purified monocytes showed minimal contamination (<2% CD3 cells) and maintained normal characteristics.
Conclusions:
- Spontaneous aggregation is a unique characteristic of human peripheral blood monocytes.
- This aggregation provides an efficient and convenient method for isolating highly purified monocytes.
- The purification process does not significantly alter monocyte morphology or function.