Lab 3 - Flow Cytometry Flashcards
(32 cards)
What is flow cytometry?
A very powerful technique that enables researchers and clinicians to analyze the properties of single cells within a population of cells
Why is flow cytometry commonly used in clinical diagnosis?
It distinguishes the presence of specific cell types or cells exhibiting specific markers
How is flow cytometry employed as both a diagnostic and prognostic tool for the analysis of lymphomas and leukemias?
Specific antibodies and fluorophores can be used to identify the presence of cell surface markers characteristic of these malignancies
What are the major areas of use for flow cytometers?
Determination of ploidy and cell cycle analysis
What basic systems do all flow cytometers have in common?
1) Fluidic system
2) Illumination source
3) Optical and electronics system
4) Data storage and computer control system
What does a fluidic system do?
Determines how light meets and interacts with cells or particles
What typically occurs in the fluidic system?
- Single particle sample suspensions are directed into a diluent (sheath fluid) and forced into a chamber by air pressure
- While the sample travels by laminar flow through the chamber, the pressure of the sheath fluid aligns the cells and/or particles in single file
What will the illumination source of the flow cytometer do?
Project a beam of laser light through the continuous liquid stream of sheath fluid buffer to illuminate cells in suspension
Why are lasers used as the illumination source?
Because the beams remain compact with little divergence, they have high spectral purity (single wavelength), and they are excellent excitation sources
What is done to cells in order to facilitate the measurements of biological properties?
Cells are often conjugated to fluorescent probes that bind to specific cellular constituents
What are the monoclonal antibodies conjugated to?
- Fluorochromes,
- Fluorescent stains (e.g., propidium iodide)
- Transgenes that are fused to a reporter (e.g., green fluorescent protein)
When are fluorescent probes excited?
As cells individually pass through the laser path at a rate as high as 1000 cells per second
What do excited fluorescent probes result in?
The emission of light at detectable wavelengths
What amount of fluorescence is emitted as a cell passes by the laser?
One that is directly proportional to the amount of fluorescent probe that is conjugated to the cell
What happens after the laser beam strikes the cell?
Light is scattered in all directions, and light emission subsequently enables the measurement of several biochemical, biophysical, and molecular properties
What happens in the optical and electronics system?
It detects light that is scattered forward, light that is scattered at 90 degrees, and the fluorescent light signals that are emitted upon excitation
What does the data storage and computer control system do?
Interprets the recorded signals and presents them as comprehensible data
What does a flow cytometer ultimately do?
Analyzes the light scattering parameters and generate data related to the structural and morphological features of a cell
What is the intensity of the low angle forward scatter proportional to?
Cellular diameter and size
What does the orthogonal or side (90 degree) intensity indicate?
The quantity of granular structures within the cellular population
What else is light scatter alone often quite useful and commonly used for?
Excluding dead cells, cell aggregates, and cellular debris from the generated fluorescence data
How will flow cytometry be used in this lab?
As a means to examine the cell cycle
What is the protocol for this lab based on?
The fact that at different stages of the cell cycle, cells have different DNA content
What kind of dye is used in this lab?
One that allows quantitation of the amount of cellular DNA - Propidium iodide (PI)
