Cellular pathology: Flow cytometry - Applications

Question 1

What is one of the main applications of flow cytometry?

Answer 1

• The analysis of cell cycle position by quantitation of cellular DNA.

Question 2

Why is flow cytometry still the method of choice for cell cycle analysis?

Answer 2

• Because it provides fast, accurate determination of cell cycle distributions.

Question 3

Describe the simplest univariate cell cycle analysis method

Answer 3

• Cellular DNA is detected using a fluorescent dye that binds preferentially to DNA.
• Propidium iodide (PI) is most commonly used fluorescent dye used

Question 4

Propidium iodide needs to cross the cell membrane in order to reach the DNA but isn’t permeable to the cell membrane. How do u make sure that the propidium iodide crosses the cell membrane?

Answer 4

• You produce holes in the plasma membrane so that the propdium iodide can pass through (permeabilization of the plasma membrane)

Question 5

The histogram below shows the results of using PI to measure the cell cycle. What does this histogram tell you about the cell cycle of this cell?

Answer 5

• Shows that there are 3 clear populations of DNA - G₀/G₁, S and G₂/M
• Also shows that the amount of DNA at G₂/M phases is double that of the G₀/G₁ phases

Question 6

How can PI be used to analyse cell viability?

Answer 6

• PI cannot normally cross the cell membrane so healthy cells won’t fluoresce when exposed to PI
• If the PI penetrates the cell membrane, it will cause that cell to fluoresce and so the cell membrane of that cell is assumed to be damaged
• Cells that are brightly fluorescent with the PI are therefore damaged or dead

Question 7

What is apoptosis?

Answer 7

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

Question 8

What are some of the characteristics of a cell underging apoptosis?

Answer 8

• Condensation of the chromatin material
• Blebbing of nuclear material
• Often accompanied by internucleosomal degradation of DNA - gives rise to distinctive ‘ladder’ pattern on DNA gel electrophoresis

Question 9

Briefely describe the different steps involved in the processes of necrosis and apoptosis

Answer 9

• Necrosis
  
  • Small blebs form and nuclear structure changes
  • Blebs then fuse and become larger - organelles aren’t present in the blebs
  • Cell membrane ruptures and releases the cell’s contents; organelles are NOT functional
• Apoptosis
  
  • ​Small blebs form
  • Nucleus breaks apart causing DNA to break into small pieces; oragnelles located in blebes.
  • Cell breaks into apoptotic bodies; organelles are still functional

Question 10

What are some of the methods for detecting apoptosis using flow cytometry?

Answer 10

• By staining with the dye PI (cells fixed) - holes need to be punched in plasma membrane for PI to eneter cell
• Phosphatidyl serine, can be detected by incubating the cells with fluorescein-labeled Annexin V, and PI (cells NOT fixed) - Phosphatidyl serine released by cell when undergoing apoptosis so it ends up oyutside the cell membrane
• By staining with 7-aminoactinomycin D, 7-AAD. (cells NOT fixed) - 7-AAD is DNA-specific and so it intercalates in G-C regions of DNA.

Question 11

When measuring apoptosis of a group of PI stained-cells what differences are there in the cell cycle compared to a group of cells where none are undergoing apoptosis?

Answer 11

• On the histogram showing the cell cycle of those cells there would be Sub-G₀ peak which may represent the apoptotic cells

Question 12

What are some of the issues of using the sub-G₀ peak as a marker of apoptotic cells?

Answer 12

• This sub-G0 peak may not actually represent apoptotic cells as it could also represent cellular debris or apoptotic bodies
• Also, even if the sub-G0 peak does actually represent apoptotic cells not all apoptotic cells contribute to the peak

Question 13

Explain how PI combined with AnnexinV-FITC can be used to distinguish between live and apoptotic cells

Answer 13

• A live cell is NEGATIVE for both PI and and AnnexinV-FITC.
  
  • This is because phosphatidyl serine is on the inside of the cell as the membrane is still intact which means that the AnnexinV-FITC can’t bind to the phosphatidyl serine and PI can’t move through the mebrane and enter the cell
• An early apoptotic cell is positive for AnnexinV-FITC but negative for PI
  
  • This is because during early apoptosis phosphatidyl serine is released from the cell to the outiside of the cell membrane meaning AnnexinV-FITC can bind to it.
  • However, during early apoptosis the cell membrane is still intact so PI stil can’t move through it and enter the cell
• A late apoptotic cell is POSITIVE for both AnnexinV-FITC and PI
  
  • ​This is because during late apoptosis the phosphatidyl serine has already been released to outside of cell membrane meaning AnnexinV-FITC can bind to it
  • Also, during late apoptosis the cell membrane is broken down meaning PI can enter the cell and bind to DNA in the nucleus

Question 14

What are some of the characteristics of the fluorochrome 7-Aminoactinomycin D (7-AAD)?

Answer 14

• Excitation wavelength - 488 nm
• Emission wavlength - 660 nm
• It’s long emission wavelength means that it can be used at the same time as other fluorochromes, e.g. FITC & PE labeled Ab for simultaneous evaluation of DNA content and 2-color immunofluorescence using only 488 nm excitation laser
• It’s DNA speicific - intercalates in G-C regions

Question 15

As well as DNA and phosphatidyl serine what other things can be used as markers of apoptosis?

Answer 15

• Caspases
• Cytochrome c
• P53

Question 16

What are some of the applications of flow cytometry?

Answer 16

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

Question 17

Explain how flow sorting works

Answer 17

• Flow sorting has same principles as flow cytometry
• Cells are present in suspension in single file; cells flow through nozzle tip surronded by sheath fluid; cells get hit by a laser which causes them to emit light; light is picked up by detectors which send data to computer
• Difference is that the nozzle tip is vibrating which causes the flow of the cells to break off into droplets
• During flow sorting you can use a computer to draw around the types of cell that you want
• When you do this whenever the machine detects your desired cell it will give that cell a charge just before it becomes a droplet
• This means that when that cell becomes a droplet the deflection plates will direct that droplet with your desired cell to a tube full of all the other desired cells it has detected previously
• Cells that aren’t the desired cells will just drop off into waste
• NOTE: can be used to collect more than one desired cell type - one desired cell type will have + charge while the other will have - charge

Question 18

What are some of the applications of flow sorting?

Answer 18

• Purfied cells sorted as a result of flow sorting can be used for:
  
  • Cell culture
  • Molecular biology
  • Biochemistry
  • Microscopy