Flow Cytometry Applications

 
The flow cytometer has been around for about 4 decades. During that time it has had a significant impact on various fields of biology and medicine, including cell-cycle studies in relation to effects of drugs and radiation, immunology, ploidy determination in cancers, and studies of cellular parameters, such as intracellular pH and Ca concentrations.

The A40 range covers all standard analytical flow cytometry applications – both prokaryotic and eukaryotic cells.

The high performance A40-MiniFCM is designed for applications requiring the highest possible light scatter and fluorescence sensitivity and excels in small particle applications such as archaea, bacteria and large virus.

Typical eukaryotic cell applications include:

• DNA content
• leukocyte classification
• anti-bodies to a variety of cellular proteins (up to 4 different simultaneously)
• apoptosis
• dead versus viable cell count
• physiological analysis such as intracellular Ca++ and other essential ions
• enzyme activities

Typical prokaryotic cell applications include:

• Microbial testing for bacteria, yeast, protozoa and (large) viruses in a wide range of samples (e.g. marine, bio-defense, food, water and environmental).
• Viability assessment (live versus dead cell count), for example using
   
  • a membrane potential indicator such as DiOC2(3)
  • a DNA binding dye excluded by healthy, intact cell membranes
    (e.g. propidium iodide)
  • an indicator of esterase activity (e.g. CFDA)
• DNA content (e.g. cell cycle analysis) using a DNA binding dye such as propidium iodide, SYTO, DAPI or combination (e.g. mithramycin and ethidium bromide)
• antibiotic susceptibility testing
• Gram staining to discriminate gram-positive bacteria from gram-negative bacteria
• Antibody labelling for cell identifications

Some typical data . . .

Light scatter resolution

Using light scatter alone, the A40 is capable of counting and distinguishing different types of microbe. For example, Lactobacillus and Staphylococcus populations can be completely resolved.

The A40’s unique optics allows incredibly small size differences to be measured. The data below shows resolution of 440nm, 480nm and 500nm latex test beads.

DNA Content

Fast, convenient nucleic acid stains are available to fluorescently label DNA so that the fluorescence is proportional to DNA content. This allows discrimination of cells from background debris and monitoring the cell cycle.

The fluorescent population of cells can be easily distinguished from background particles which do not contain DNA, and from particles with a different amount of DNA.

Flow cytometry using DNA staining is an essential tool for the diagnosis of certain cancers (cancer cells often have an aberrant DNA content) and for performing cell cycle analysis. The principles apply to bacteria and archaea as well as mammalian cells. Analysis of the DNA content provides information on the proliferation of the cells, in particular on the duration of the different cell cycle phases by determining the relative number of cells in the different cell cycle phases.

Viability Staining

Propidium iodide (red fluorescence – FL3 detector) and a cell permeable nucleic acid stain (e.g. with green fluorescence – FL1 detector) can be used in combination to distinguish live and dead cell populations.

Here is a mixture of live and dead (heat treated) bakers yeast. Cells with intact membrane exclude the propidium iodide and therefore exhibit very little red fluorescence, but do exhibit green fluorescence.

Dead cells with damaged cell membrane exhibit both green and red (PI) fluorescence. The same method can be applied to bacteria.

Metabolic Activity

CFDA (carboxyfluorescein diacetate) can be used to give a viability evaluation by monitoring metabolic activity. Upon hydrolysis by intracellular nonspecific esterases, CFDA forms carboxyfluoresceinesterase and this emits green fluorescence when excited by a blue laser. Green fluorescence is collected by the flow cytometer’s FL1 detector.

The cytogram shows Lactococcus prepared using

• propidium iodide to label cells with a damaged membrane, and
• CFDA to label metabolizing cells