Stepwise isolation of human peripheral erythrocytes, T lymphocytes, and monocytes for blood cell proteomics.

ABSTRACT

PURPOSE: Density gradient centrifugation and magnetic- or fluorescence-activated cell sorting are common and robust techniques for the isolation of different types of blood cells. In this article, we give detailed description of a stepwise application of these methods as one isolation strategy for enrichment of different cell types from one blood sample. EXPERIMENTAL DESIGN: The workflow targeted erythrocytes, monocytes, and T lymphocytes. Pancoll® density gradient centrifugation was used together with subsequent MACS™ isolation. Purity of monocytes and T lymphocytes was controlled by fluorescence-activated cell sorting analysis, and cells were used for carrier-ampholine-based 2D-PAGE to confirm compatibility of the procedure to standard proteomic applications. RESULTS: Gradient centrifugation resulted in an average of 125 µL of packed erythrocytes per milliliter blood. MACS™ sorting reached purities of 90 ± 2% (monocytes) and 93 ± 2% (T lymphocytes), with an average yield of 12 × 10(4) monocytes or T lymphocytes. 2D-PAGE of isolated cells showed well-separated spot patterns. CONCLUSIONS AND CLINICAL RELEVANCE A combined isolation holds substantial advantages especially in clinical studies, as it allows for the comparison of findings not only between individuals, but also between different cell types derived from one donor. Our approach ensured high reproducibility, yields, and purities of cells as required for reliable proteome analysis.