Flow Cytometry Applied to the Animal Kingdom in Studies of Natural Resource Science

Science Center Objects

Flow cytometry is a technique for rapidly analyzing large numbers of animal cells using light-scattering, fluorescence, and absorbance measurements.

Flow Cytometry
Figure 1. Basic operation of flow cytometry

The Science Issue and Relevance: Flow cytometry is a technique for rapidly analyzing large numbers of animal cells using light-scattering, fluorescence, and absorbance measurements. The robustness of this method comes from the wide range of cellular parameters that can be analyzed and the variety of science questions it can answer. Utility of this technique is abundance and distribution of a wide variety of biota can be analyzed.

Methodology for Addressing the Issue: Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths. The fluorescence can be measured to determine various properties of single particles, which are usually cells. Up to thousands of particles per second can be analyzed as they pass through the liquid stream. Examples of the properties measured include the particle’s relative granularity, size and fluorescence intensity as well as its internal complexity. An optical-to-electronic coupling system is used to record the way in which the particle emits fluorescence and scatters incident light from the laser.

Fig. 2. Dots outside the gates represent nuclei outside the main population, so contain fragmented DNA.
Figure 2. Dots outside the gates represent nuclei outside the main population, so contain fragmented DNA.

Basic operation involves creating a cell suspension which is stained with specific dyes, often fluorochromes. Then (see Fig. 1) (1) cells are drawn in liquid buffer under pressure into a (2) focused, single file stream that flows across the path of a laser beam which excites the dye, causing it to (3) emit light wavelengths that are (4) detected and (5) displayed as multiparametric data.

Detectable Parameters of Cells:

  • cell size
  • complexity of cell contents

  • cell count
  • DNA content and fragmentation (Fig. 2)

  • mitochondrial function

  • cell membrane integrity

  • protein presence

Some Attributes of Flow Cytometry Technology:

  • Speed (e.g., from 300 cells per second to 3000 cells per second)

  • Large sample size (n=10K – 100K/tube)

  • Accuracy (e.g., rare event identification)

  • Living or preserved cells

  • Portability to the field

Some applications in environmental research at WARC (see also Fig. 3 below):

  • Endocrine disruption studies: Sperm quality of male fish from across the U.S. (e.g., largescale suckers, razorback sucker, yellow perch, small mouth bass, pallid sturgeon)

  • Species differentiation: Endangered Pallid vs Shovelnose vs Pallid/Shovelnose sturgeon hybrids

  • Deepwater Horizon: Gulf Sturgeon condition pre- and post- oil spill

  • Invasive Asian Carp: Determining if carp caught in the wild are diploid (reproductively capable) or triploid (functionally sterile)

Fig. 3 - Phylogenetic tree displaying species studied by flow cytometry in the WARC laboratory.
Figure 3. Phylogenetic tree displaying species studied by flow cytometry in the WARC laboratory.

Future Steps: Flow cytometry has many applications in future WARC research and is its use is dependent on proposal acceptance and funding.