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Flow Cytometery Applications

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

The high performance A40-Microbiology 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

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.

Apogee is leading the field in reliable light scatter & fluorescence measurements of virus particles, making it easy to count many viruses in a research laboratory.

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Application Note Downloads


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.

Resolving Lactobacillus and Staphylococcus

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.

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.

Archaea analysis

Chicken erythrocytes with their DNA stained with Propidium Iodide:

Chicken erythrocytes with their DNA stained with Propidium Iodide

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.

Live / dead bakers yeast analysis

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

Lactococcus

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 A40-Microbiology system in such applications, even for a relatively dilute sample.

Marsilia Aquatic Fern 2

News

New Apogee Software

Keep up to date: Contact us now to get release v2-02 for free!


Dakar Conference 4ème Cours de Rétrovirologie biologique, Dakar (26th Oct - 24th Nov 2009)

This year, new Auto40 features will be presented by Mr Languillat of Inodex, Paris.

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8th UK Meeting on Microbiology, Genetics and Molecular Biology

Apogee sponsored this meeting at the University of Nottingham, January 9th 2009.

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Dual laser excitation - January 2009

Apogee has delivered its first dual laser flow cytometer with 405nm and 488nm laser.

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Sénégal 15th ICASA
 
Dakar,
Sénégal

3rd-7th December 2008

Apogee exhibited its Auto40 model at ICASA in Dakar

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Apogee at 3eme Cours de Retrovirologie Biologique

In collaboration with Inodex (France), Apogee exhibited and demonstrated the new 'Auto40' flow cytometer...

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New Telephone Numbers

Apogee has a new telephone system. Please note our new phone numbers:

• Manufacturing:
  020 8123 6824

• Sales and Technical Support:
  020 8123 6831


NEW Low Cost Auto40

Full function, high quality flow cytometer with automated data analysis.

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Marine Microbiology:

North-Western Mediterranean Sea Cruise.

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New Data Acquisition and Analysis Software (version 1-88)

Ability to overlay data from multiple files.

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Dakar, Senegal

Apogee presented the A40-Microbiology to students . . .

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Cytometry 2006, Bruges

Thank you to all who visited our booth at 'Cytometry 2006'...

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Special Offer to African A40 Users

If you have an A40 flow cytometer in Africa, contact us now quoting this website offer to receive a free software upgrade to version 1-80 ...

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EMBO Workshop, Carlsberg Academy, Copenhagen

"Cell Cycle & Cytoskeletal Elements in Bacteria"

Apogee sponsored this popular and successful meeting and displayed the high sensitivity of the A40-Microbiology ...

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