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October 01, 2009 (Vol. 29, No. 17)

Cell-Counting Technologies Are Evolving

Flow Cytometer Aims to Improve Upon Traditional Methodologies

  • Click Image To Enlarge +
    Figure 1. The Accuri C6 Flow Cytometer System

    Technology that gives researchers the ability to quickly and precisely determine the number of cells with a given phenotype in cell cultures or fresh tissue samples is of great interest in diverse fields, including clinical research and diagnostics, drug discovery, and molecular biology. In this article, we present information on the function and use of the Accuri® C6 Flow Cytometer® System from Accuri Cytometers, which can quickly and reproducibly provide single-platform cell counts on fluorescently labeled subsets in mixed cell populations.

    For years the combination of a light microscope and hemacytometer have been the tools of choice for performing cell counts in life science research laboratories. This method is slow and prone to error. When hemacytometer counts and flow-cytometric phenotypic data are combined to determine cell subset numbers, errors are multiplied. Digital flow cytometers, utilizing laminar-flow fluidics, allow fast, phenotypic data collection (at rates up to 10,000 events per second) on a wide range of cell types (submicron-sized bacteria through large mammalian cell lines), but still require the addition of counting beads to each sample to calculate cell-subset concentration.

    On the other hand, flow cytometers with syringe-driven fluidics can deliver absolute count measurements without the addition of counting beads to samples, but are often limited by lower data acquisition rates (<1,000 events per second), diminished fluorescence and light-scatter resolution, and a propensity for flow-cell clogging.

    The Accuri C6 Flow Cytometer System is a small flow cytometer that has a unique peristaltic-pump-driven, laminar-flow fluidics system (Figure 1). The C6 combines the advantages of hydrodynamically focused cell sampling (high data-acquisition rates, good light scatter and fluorescence resolution) with the ability to report absolute counts for any identified population in a sample.

    Two applications of direct cell-concentration determination with the C6 are presented here: viability assessment of cultured cell lines and platelet counts for whole, human peripheral blood samples. Both methods make use of the fluorescence and light-scatter measurements possible with a flow cytometer to identify sub-populations of interest. Cell-counting data collected by at least one other traditional method (counting beads or hemacytometer) is included for comparison.

  • Instrumentation

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    Figure 2. Viable cell-count determinations with the C6: (A) Setting viable-cell gate (P5). (B) Setting dead-cell gate (P6). (C) CFlow Software Statistics Table. (D) Comparison of mean viable cell count.

    The C6 was validated, before data collection, for accurate volume measurement using Accuri Volume Validation Beads, fluorescent beads prediluted at a known concentration.

    CFlow® Plus software was used for cell-count data collection. A fluidics setting of medium (flow rate=35 µL/min, core size= 16 µm) was used during sample collection.

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