Flow Cytometry Applications

Question 1

1. What is one of the earliest applications of flow cytometry?

Answer 1

Analysis of cell cycle position by quantitation of cellular DNA

Question 2

2. What is the method of choice for cell cycle distribution? and why?

Answer 2

Flow cytometry

-Fast and Accurate

Question 3

3. What is the univariate cell cycle method?

Answer 3

o Cellular DNA is detected using a fluorescent dye that binds preferentially/specifically to DNA.

Question 4

4. What is the most common fluorescent dye?

Answer 4

oPropidium iodide (most commonly used) undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilization(punching holes in the membrane) to allow PI to enter

Question 5

5. What nm does propidium Iodide emit at?

Answer 5

600/620

Question 6

6. In the normal cell cycle, what is intensity directly proportional to?

Answer 6

The amount of DNA

We can use PI to quantify the cells in each phase of the cell cycle

Question 7

7. Okay so the PI assay for Cell viability basically tells us whether cells are damaged or dead, how does it do this?

Answer 7

o PI cannot normally cross the cell membrane in a normal, viable cell.
o If the PI penetrates the cell membrane, it is assumed to be damaged - cells that are brightly fluorescent with the PI are damaged or dead.

Question 8

8. We know from previous lectures that apoptosis is programmed cell death , what is characterised by?

Answer 8

• Chromatin Condensation

- Blebbing (outward protrusions) of nuclear material

Question 9

9. Apoptosis is often accompanied by internucleosomal degradation, how would you identify this on a DNA gel electropheresis?

Answer 9

Distinctive “Ladder” pattern

Question 10

10. There are three main methods to detect Apoptosis , explain the method that involves staining with the dye PI?

Answer 10

• Cells fixed
• Fluorescence intensity on x axis
• Sub G0 peak is seen and this is where apoptotic cells will appear

Question 11

11. What is evidence for and against the PI staining method for detecting apoptosis?

Answer 11

o Evidence against: Because not all apoptotic cells show this peak, it’s argued it may be debris rather than cells.

o Evidence for: When an apoptotic stimulus is added, the peak increases in size

Question 12

12. What is the method of detecting apoptosis using

Phosphatidyl Serine

Answer 12

Phosphatidyl serine can be detected by incubating the cells with fluorescein labeled Annexin V and PI which bind to phosphatidyl serine (cells not fixed).
o In live cells, phosphatidyl serine (PS) is on the inside of the cell so the Annexin can’t bind to it and because the membrane is not damaged, PI can’t enter.
o In early apoptotic cells, PS is on the outside of the cell so Annexin can bind to it however PI still can’t enter.
o In late apoptotic/dead cells, Annexin binds to PS and PI enters the cell due to a damaged surface membrane

Question 13

13. How do we quantify how many apoptotic cells there are using Phosphatidyl Serine ?

Answer 13

Using a dye graph:
- Draw a graph with PI ( on y axis) and Annexin C FTC ( on the x axis)
• Live cells are negative for both dyes and dead cells are positive for both dyes.
• Apoptotic cells are Annexin positive but PI negative

Question 14

14. How do we test for apoptosis using 7-AAD?

Answer 14

3. By staining with 7-aminoactinomycin D aka 7-AAD (cells not fixed).
   o Excitation occurs at ~488 nm and Emittance at: ~660 nm.
   o DNA-specific: intercalates in G-C regions
   o Long emission wavelength: can be used with FITC & PE labeled Ab for simultaneous evaluation of DNA content and 2-colour immunofluorescence (using only 488 nm Ex).
   o 7AAD staining of bone marrow:
   • Live cells stain brightly (90.0%).
   • Apoptotic cells stain lightly/faintly (6.3%).

Question 15

15.What are some other methods for detecting apoptosis - not staining/dye’s?

Answer 15

Can measure/quantitate :
Caspases
Cytochrome C release
Parts of the intrinsic and extrinsic apoptotic pathways.

Question 16

16. What are some applications of apoptosis detection?

Answer 16

• Immunophenotyping of leukaemias & lymphomas
• Detection of Minimal Residual Diseases (MRD).
• Stem cell enumeration (rare cells that can be quantitated relatively easily/quick).
• CD4/CD8 in HIV
• Measurement of intracellular cytokines and cell proliferation (dyes staining plasma membrane reduce as plasma membrane divides for daughter cells).
• Study of cell cycle, viability & apoptosis
• Assessment of transfection efficiency

Question 17

17. What is cell sorting?

Answer 17

Cell sorting is the process of taking cells from an organism and separating them according to their type.

Question 18

18. What is the process of cell sorting?

Answer 18

1. A specific region is selected on the computer.
2. The machine vibrates the nozzle to charge the stream so that the specific cells that satisfy the region that are in flow can be broken off into droplets.
3. The drop containing the cell is then pulled away and separated into tubes/plates by the deflection plates.
4. The computer then removes the charge from the stream so that cells continue to stay in flow until another cell that fits the criteria comes along.

Question 19

19. What kind on environment is cell sorting carried out in?

Answer 19

carried out in a sterile environment so they can be cultured directly.

Question 20

20. What is pre-sort process?

Answer 20

o Pre-sort: the analysis of the impure cells before cell sorting which allows the user to set parameters on the machine for the separation.

Question 21

21. What is the post-sort process?

Answer 21

the analysis of the purified cells (should be around 90% pure)