Immunology: Fluorescence-Activated Cell Sorting (FACS)

Flow cytometry is designed to determine the relative proportions of CD4+ and CD8+ T cells, B cells, NK cells, and monocytes in the mononuclear cell population. A Becton-Dickinson FACSCalibur 3-laser FACS machine was used to assess immune status. This machine records CD4+/CD8-, CD8+/CD4-, NK, B cell, and monocyte numbers, in addition to the CD4+/CD8+ ratio. Numerous mouse KO lines have been shown to display aberrant levels of different cell populations in this assay, including the TPO, CD18, and RAG-1 knockouts (Bunting et al., 1997; Weinmann et al., 2003; Mombaerts et al., 1992).

The mononuclear cell profile is derived by staining a single sample of lysed peripheral blood from each mouse with a panel of six lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8 FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodies stain mutually exclusive cell types. The samples are analyzed using a Becton Dickinson FACSCalibur flow cytometer with CellQuest software.

Displayed below is a sample graph of how FACS results are presented. In comprehensive phenotypic data packages graphs are interactive. Raw or calculated data and statistics can be seen by clicking on points in the graph.

Figure illustrates number of TCRΒ+ cells in mutant mice. Percentage of CD4+ and CD8- cells (left), CD8+ and CD4- cells (center) in peripheral blood mononuclear cells, and CD4+/CD8+ ratio (right) of wild type littermates (green circle), heterozygous (blue triangle), homozygous (red diamond), and recent historical wild type (purple line) mice are plotted against long-term historical values (± 2 standard deviations) for wild type animals (green shading). Recent wild type values are calculated from data collected within 60 days of current measures and long-term historical values are derived from data collected on more than 10,000 wild type mice.

Figure illustrates number of TCRΒ- cells in mutant mice. Percentage of NK cells (left), B cells (center), and monocytes (right) in peripheral blood mononuclear cells of wild type littermates (green circle), heterozygous (blue triangle), homozygous (red diamond), and recent historical wild type (purple line) mice are plotted against long-term historical values (± 2 standard deviations) for wild type animals (green shading). Recent wild type values are calculated from data collected within 60 days of current measures and long-term historical values are derived from data collected on more than 10,000 wild type mice.