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SIMULTANEOUSLY MEASURE CELL PROLIFERATION AND STUDY PHENOTYPICALLY DISTINCT SUBSETS OF A MIXED CELL POPULATION.

Using flow cytometry, the Cell Census Plus^TM; System simultaneously measures cell proliferation of phenotypically distinct subsets of cells within a heterogeneous population. Cell Census Plus contains a red fluorescent dye to label the cell’s membrane; microbeads to standardize instrument setup and normalize cell counts; and software, the cell proliferation model, to interpret the histogram data.

Provides the number of cell divisions (up to ten generations), relative abundance of each daughter generation, the fraction of non-proliferating cells and the proliferation index.

PLUS
study distinct cell subsets within a total cell population via immunophenotyping (i.e., antibodies to cell surface markers).

Measures the endpoint of multistep activation, instead of discrete, early events, such as up-regulation of CD69.
Provides for standardized cytometer setup and normalized cell counts.
Works with ModFit, a computer program, to deconvolute histograms.
Labels all cell types with a universal fluorescent membrane dye. Membrane labeling is fast, and sufficient for kinetic studies
Does not interfere with biological and proliferative activities of cells.

Uses nonradioactive reagents.

Figure 1a and 1b
Histogram of PKH26-labeled cells on day zero (a) and histogram of PKH26-labeled cells on day 4 of a mitogen stimulated culture (b). Human peripheral blood mononuclear cells, isolated from normal blood, were labeled with PKH26 membrane dye. A sample of cells was fixed and analyzed as the day zero control (Figure 1a). The remaining cells were stimulated with 2.5 µg/ml PHA in RPMI plus 10% fetal bovine serum for 4 days. Prior to flow cytometric analysis, cells were immuno-stained with FITC-CD4 and Quantum RedTM-CD45RO. List mode data were collected on a FACScan cytometer using Lysis II software. The total population, gated by light scatter, was analyzed for proliferation using Cell Census Plus. The open histogram represents the raw data. The shaded peaks are the result of histogram deconvolution. Each shaded peak in Figure 1b represents a daughter generation.