Flow Cytometry applications Flashcards
What is flow cytometry still the method of choice for?
• Flow cytometry is still the method of choice for fast, accurate determination of cell cycle distributions
What is cellular DNA detected using in the simplest method?
• In the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA
What is the most commonly used fluorochrome?
Most commonly used fluorochrome is Propidium Iodide
What does propidium iodide require in order to allow propidium iodide to enter?
○ It requires permeabilisation of the plasma membrane to allow the PI to enter (punching holes in the membrane) (AKA fixing)
Can propidium iodide be used with other fluorochromes?
○ PI can be used simultaneously with other fluorochromes
What does PI allow?
• PI allows quantification of the proportion of cells at each stage in the cell cycle
How does the assay for PI-cell viability work?
How the assay works:
○ This assay works to assess viability of the cell and can tell you whether the cell is damaged
§ 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 damaged or dead
What are the characteristics of apoptosis?
- 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
Apoptosis vs necrosis
Necrosis is messy whereas apoptosis is neat and orderly
What are the 3 detection methods for apoptosis?
- Stain cells with PI
- Phosphatidyl serine
- Staining with 7-aminoactinomycin D
Stain cells with PI
○ Cells fixed ○ Fluorescence intensity on x axis ○ Sub G0 peak is seen and this is where apoptotic cells will appear ○ Disadvantage: not reliable -People argue that
How can phosphatidylserine be detected and why?
○ Can be detected by incubating the cells with fluorescein-labeled Annexin V, and PI (cells not fixed)
○ Dead cells have PS on the outside whereas healthy cells have this inside
§ Dead cells therefore can be quantified
Annexin cannot bind to live cells as PS is on inside
What happens to PS when cell is in early apoptosis and hence what will the cells appear positive and negative for?
○ When cell is in early apoptosis, the PS is flipped towards the outside
§ Cells will therefore appear positive for annexinV-FITC
§Will be negative for PI however as the membrane is still intact
What happens to PS in late apoptosis and whats it positive for?
○ Late apoptosis
§ Positive for PS
§ Also now positive for AnnexinV-FITC because membrane is disintegrating
Staining with 7-aminoactinomycin
- Cells not fixed
- One step method
