Flow Cytometry - Applications

Question 1

Describe one of the applications of flow cytometry.

Answer 1

One of the earliest applications of flow cytometry was the analysis of cell cycle position by quantitation of cellular DNA.

Flow cytometry is still the method of choice for fast, accurate determination of cell cycle distributions.

Question 2

Describe the simplest univariate cell cycle method.

Answer 2

In the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA.

Propidium iodide is most commonly used. It undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilization of the plasma membrane.

Question 3

How is PI (propidium idiodide) used in assessing cell viability?

Answer 3

How the assay works:

• PI cannot normally cross the cell membrane.
• If the PI penetrates the cell membrane, it is assumed to be damaged.
• Cells that are brightly fluorescent with the PI are considered damaged or dead.

Question 4

What is apoptosis, and what are some of its characteristics?

Answer 4

Apoptosis is programmed cell death where the cell goes through a highly regulated process of “dying”.

Characteristics are:

• condensation of the chromatin material
• blebbing of nuclear material
• often accompanied by internucleosomal degradation of DNA giving rise to distinctive ‘ladder’ pattern on DNA gel electrophoresis

Question 5

Describe the different steps between necrosis and apoptosis.

Answer 5

NECROSIS:

• small blebs form; the structure of the nucleus changes
• the blebs fuse and become larger; no organelles are located in the blebs
• the cell membrane ruptures and release the cell’s contents; the organelles are not functional

APOPTOSIS:

• small blebs form
• the nucleus begins to break apart, and the DNA breaks into small pieces; the organelles are also located in the blebs
• the cell breaks into several apoptotic bodies; the organelles are still functional

Question 6

What are some detection methods for apoptosis?

Answer 6

• by staining with the dye PI (cells fixed)
• phosphatidyl serine, can be detected by incubating the cells with fluorescein-labeled Annexin V, and PI (cells not fixed)
• by staining with 7-aminoactinomycin D (cells not fixed)

Question 7

How can you distinguish different populations of cells using PI and AnnexinV?

Answer 7

If the cell doesn’t show active AnnexinV or PI, it’s a live cell.

If the cell shows AnnexinV but not PI, it’s an early apoptotic cell.

If the cell shows both AnnexinV and PI, it’s a late apoptotic/ necrotic cell.

Question 8

Describe 7-Aminoactinomycin D (7-AAD).

Answer 8

Ex: ~488 nm
Em: ~660 nm

It’s DNA-specific, so it intercalates in G-C regions.
It also has a long emission wavelength; we can use it with FITC & PE labeled Ab for simultaneous evaluation of DNA content and 2-color immunofluorescence using only 488 nm Ex.

Question 9

List some applications of flow cytometry.

Answer 9

• Immunophenotyping of leukaemias & lymphomas
• Detection of MRD
• Stem cell enumeration
  CD4/CD8 in HIV
• Measurement of intracellular cytokines
• Study of cell cycle, viability & apoptosis
• Measurement of cell proliferation
• Assessment of transfection efficiency

Question 10

Describe the fine-tuning of cell sorting.

Answer 10

The basics of flow cytometry are present: the cells are in a suspension in a single file, a laser is put on them, causing them to emit light that is a picked up and tells a computer connected to it what type of cell it is.

In cell sorting, we can draw a range around the types of cells we want, so the computer will ‘sort them out’.

The nozzle tip is always vibrating, so much that the stream breaks off into droplets at some point. Within milliseconds of detection of a certain cell, if it is the type that we want, the computer will charge that cell when it is at the end of a droplet, breaking it off.

The cell is then collected into a tube by being pulled towards a deflection plate (due to its charge). The rest of the cells the go to be collected for waste.

As a result, we get a sample of very purified cells, with endless possibilities in research.