Flow Cytometry Applications Flashcards
- What is one of the earliest applications of flow cytometry?
Analysis of cell cycle position by quantitation of cellular DNA
- What is the method of choice for cell cycle distribution? and why?
Flow cytometry
-Fast and Accurate
- What is the univariate cell cycle method?
o Cellular DNA is detected using a fluorescent dye that binds preferentially/specifically to DNA.
- What is the most common fluorescent dye?
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
- What nm does propidium Iodide emit at?
600/620
- In the normal cell cycle, what is intensity directly proportional to?
The amount of DNA
We can use PI to quantify the cells in each phase of the cell cycle
- Okay so the PI assay for Cell viability basically tells us whether cells are damaged or dead, how does it do this?
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.
- We know from previous lectures that apoptosis is programmed cell death , what is characterised by?
- Chromatin Condensation
- Blebbing (outward protrusions) of nuclear material
- Apoptosis is often accompanied by internucleosomal degradation, how would you identify this on a DNA gel electropheresis?
Distinctive “Ladder” pattern
- There are three main methods to detect Apoptosis , explain the method that involves staining with the dye PI?
- Cells fixed
- Fluorescence intensity on x axis
- Sub G0 peak is seen and this is where apoptotic cells will appear
- What is evidence for and against the PI staining method for detecting apoptosis?
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
- What is the method of detecting apoptosis using
Phosphatidyl Serine
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
- How do we quantify how many apoptotic cells there are using Phosphatidyl Serine ?
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
- How do we test for apoptosis using 7-AAD?
- 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%).
15.What are some other methods for detecting apoptosis - not staining/dye’s?
Can measure/quantitate :
Caspases
Cytochrome C release
Parts of the intrinsic and extrinsic apoptotic pathways.
