Flow Cytometry Applications

Apogee 'Universal' and 'Micro' flow cytometers cover virtually any analytical application. The high performance A50-Micro model is in a class of its own for:

• microparticle (microvesicle) flow cytometry
• picoplankton (0.2-2.0µm) & nanoplankton (2-20µm)
• bacteria and archaea
• large viruses

Microvesicles / Microparticles

Apogee's Micro model is ideal for measuring a wider range of microparticles than is possible on conventional flow cytometers. Quite simply, its exceptional sensitivity at multiple light scattering angle ranges allows it to resolve smaller vesicles. Microparticles range in size from about 100nm to 1000nm, but conventional flow cytometers are limited to analysing the top half of this range by light scatter (microvesicles scatter less than corresponding latex beads). In combination with data from its fluorescence detectors, the Micro model offers unique performance in this cutting edge field of research.

Apogee's A50-Micro is more sensitive than the earlier generation A40-Micro, but recent publications demonstrate the excellent performance of even the older A40 generation. For example:

"A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer."
Journal of Thrombosis and Haemostasis 2011 Jun;9(6):1216-24
Chandler, W.; Yeung, Wandy; Tait, Jonathan

Typical eukaryotic cell applications ('Micro' or 'Universal' models):

• DNA content
• Leukocyte phenotyping
• Apoptosis
• Dead versus viable cell count
• Physiological analysis such as intracellular Ca++ and other essential ions
• Enzyme activity
• Multiple cellular protein measurements (e.g. up to 4 antibodies simultaneously)

Typical prokaryotic cell applications ('Micro' model):

• Microbial testing for bacteria, yeast, protozoa and (large) viruses in a wide range of samples (bio-defence, food, water and environmental)
• Marine microbiology (e.g. picoplankton and nanoplankton)
• 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

NEW Light Scatter Size Calibration Kit

Light scatter size calibration method for submicron particles based on the Mie solution to Maxwell's equations. Refractive index considerations are essential when estimating particle size from light scatter data. Apogee's revolutionary method provides an accurate particle size derived from the wavelength of light and refractive index of the particle.

A fantastic new technology for the standardization of light scatter measurements: Ideal for sizing bacteria, microvesicles / cell derived microparticles, liposomes, lipoproteins, ectosomes, endosomes...

» Contact us now for further information

Flow Cytometry of Viruses

Large viruses have been measured by flow cytometers before, typically using an RNA or DNA stain, but useful light scatter information from individual virus particles is difficult to obtain due to their size. The A50-Micro leads the field in light scatter & fluorescence measurements of virus particles, making it easier to count large viruses in a research laboratory.

» Contact us for more information.

Some typical data . . .

Light scatter resolution

Using light scatter alone, the A50 is capable of counting and distinguishing different types of microbe. For example, Lactobacillus and Staphylococcus populations can be completely resolved.

The A50'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.

Chicken erythrocytes with their DNA stained with Propidium Iodide:

» Download Application Note - DNA Content & Ploidy Analysis

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

Ploidy Analysis of Plant Cells

Flow cytometry is established as a useful, quick method to determine efficiently, reproducibly and at a reduced cost per sample, the relative nuclear DNA content and ploidy level of a large number of species including both plants and animals.

For example, Marsilea is a species of aquatic ferns. The Marsilea data below demonstrates the excellent CV (resolution) of the A50-Microbiology system in such applications, even for a relatively dilute sample.

» Download Application Note - DNA Content & Ploidy Analysis

Application Notes

Please contact Apogee for application support notes, explaining which applications you are interested in: