Beyond six colors: A new era in flow cytometry

• DOI: 10.1038/nm0103-112
• Publication Date: 2003-01-03T19:13:06Z
• Authors: Stephen C. De Rosa; Jason M. Brenchley; Mario Roederer
• Keywords: 0303 health sciences; Lasers; Color; History, 20th Century; Flow Cytometry; 3. Good health; Mice; 03 medical and health sciences; Phenotype; T-Lymphocyte Subsets; Animals; Humans; Lymphocytes; Biomarkers; Spleen; Fluorescent Dyes

+T cells’. Instead, investigation of ‘fine’ lymphocyte subsets is necessary to understand the mechanisms of immunological disease and protection. To distinguish among these specific cell types, it is necessary to measure simultaneously as many as six different T-cell-associated ‘markers’ (typically cell surface proteins); further functional characterization requires the additional measurement of responses (such as cytokine production, proliferative capacity or apoptotic potential). This has necessitated the development of flow cytometric technologies capable of detecting considerably more parameters than the typical 3- or 4-color cytometers prevalent to date. Current technology is capable of measuring 2 scatter and 12 fluorescence parameters simultaneously and individually for each cell. Because this technology comes with unique problems (and solutions), we term it polychromatic flow cytometry (PFC), which we use to refer in general to flow cytometric analyses encompassing 6 or more colors. Instrument manufacturers have recognized the growing demand for PFC, and most now provide the hardware necessary to carry out these types of measurements. During the past few years, several research groups have employed custom-built instruments with the capacity to quantify as many as 12 fluorescences. In this paper we explore how this multicolor cytometric technology is substantially refining extant methods to allow new experimental designs. Together, these types of studies are providing a completely new view of the immune system that encompasses a far greater heterogeneity of cell type and function than previously imagined.