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.

Methodology for Addressing the Issue: Basic operation involves creating a cell suspension which is stained with specific dyes, often fluorochromes. Then, as shown in Figure 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. An example of a cytogram showing nuclei in whole blood stained with a fluorescent dye is Figure 2.

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

Figure 2. This is an example of a cytogram from a flow cytometric analysis of blood cell nuclei from a Gulf sturgeon that had been exposed to an oil spill. The dots outside the gates represent nuclei outside the main population, demonstrating fragmented DNA.  Fragmented DNA indicates that a genotoxic event likely occurred.

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
  • Etc.

 

Some Attributes of Flow Cytometry Technology:

  • Speed (e.g., from 300 cells- 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

 

Future Steps: This technique will continue to be applied to environmental research at WARC (see Figure 3), for example:

  • 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, fathead minnows, mosquitofish)
  • Species differentiation: Endangered Pallid vs. Shovelnose vs. Pallid/Shovelnose hybrids
  • Genetic sex determination: Phenotypically similar individuals of unknown sex can be distinguished (e.g., king rails)
  • Sperm viability testing: Multiple fish and amphibian species
  • Genome size determinations and hybrid identification: Multiple species
  • Deepwater Horizon: Gulf sturgeon condition pre- and post-oil spill
  • Invasive Carp: Determining if carp caught in the wild are diploid (reproductively capable) or triploid (functionally sterile); predicting and monitoring spread, as well as source tracking.

 

phylogenetic tree

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