FLOW CYTOMETRY - INTRODUCTION AND APPLICATIONS Flashcards
What is Flow Cytometry?
Technique which simultaneously measures several physical characteristics belonging to a SINGLE CELL in SUSPENSION This is done by LIGHT SCATTER and FLUORESCENCE Essentially, flow cytometry allows us to Measure properties of cells in flow. But to go more detailed and separate cells on these properties, it is known as Cell sorting: Sorting (separating) cells based on properties measured in flow Also called Fluorescence-Activated Cell Sorting (FACS)
What can a flow Cytometer tell us about a cell?
Its Relative Size Its Relative Granularity/Internal Complexity Its Relative Fluorescence Intensity
As well as flow cytometry as a method of visualisation, what other method can be used and how does it differ from flow cytometry?
You can also use Fluorescence Microscopy. Fluorescence Microscopy analyses about 20 or so cells at a time so would need to scan across etc whereas flow cytometry is already more advanced as it can scan hundreds in one go so is more quantitative. Also if you were looking for rare cells, would be much much harder/impossible to do so with Fluorescence Microscopy but you can with flow cytometry. Also, Fluorescence Microscopy is more subjective as you decide the brightness by eye whereas flow cytometry does that for you-it quantifies the fluorescence. (so more accurate when it comes to comparison)
What are the basics of flow cytometry?
Can be split into 3: 1.Fluidics: 2.Optics 3.Electonics Cells in suspension flow in single-file through an illuminated volume where they scatter light and emit fluorescence that is collected, filtered and converted to digital values that are stored on a computer Fluidics- cells in suspension flowing in single file Optics- hit by a laser and scatter light and filtered Electronics-light signals are converted to digital signals that can be stored on a computer and re visited later to be stored on a computer.
What happens in the first stage of Flow Cytometry - Fluidics?
Need to have cells in suspension flow in single file Accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice Sample fluid flows in a central core that does not mix with the sheath fluid - Laminar flow Introduction of a large volume into a small volume - Hydrodynamic Focusing
How do the cells interact with a laser in the optics stage of flow cytometry?
Lasers Single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths can provide from milliwatts to watts of light can be inexpensive, air-cooled units or expensive, water-cooled units provide coherent light (Single frequency) laser hits the cells which are moving through most common wavelength of laser used is 488nm. once light hits the cell, it is scattered in proportion to the size of the cell (known as forward light scatter) the laser light is also emitted at a right angle to the cell (light hitting proportional to granularity(known as side scatter).
Then the signals are processed from the detectors and converted into ways which will be useful for analysis.
When does fluorescence happen?
What is Stokes shift?
Fluroescence happens when a laser hits a fluorochrome and it is excited at one wavelength, then when the fluorochrome goes back to it unexcited state, it emits fluorescence at a higher wavelength. (see image attached for most common one)
Stokes shift is the energy difference between the lowest energy peak of absorbence and the highest energy of emission- essentially the difference between the above.
Name some examples of fluorochromes and their dyes (colours)
most commonly:
Fluorescein isothiocyanate (FITC) GREEN
(excited at 488 and emits at around 520 nm)
also:
Phycoerythrin (PE) ORANGE
(emits further out at about 580-in the orange zone)
Peridinin Chlorophyll Protein RED
(PerCP)
(emits even further out at 620-in the red zone)
all the above are excited at 488
In flow cytometry, you need cells in suspension, what are some examples of some ideal cell types?
Peripheral blood
Bone marrow
Fine Needle Aspirate
CSF and other fluids
Fresh Tissue
What are the 2 methods of labelling cells with monoclonal antibodies and fluorochromes?
DIRECT : Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes
INDIRECT: Unconjugated MoAbs
In direct, the fluorochrome is conjugated onto the antibody which binds directly onto the antigen.
Whereas, in indirect, you label with monoclonal and coming in with an antibody to the monoclonal that has the fluorochrome attached.
You can plot these on histograms and dot plots.
What fluorescent dye is most commonly used to detect cellular DNA?
- In the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA.
- Propidium iodide is most commonly used. It undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilization of the plasma membrane.
How does an assay work when using PI to check for if a cell is damaged etc?
Using Propidium Iodide(PI)
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 is apoptosis? What are some characteristics of cells when this occurs?
Apoptosis is programmed cell death where the cell goes through a highly regulated process of “dying”
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
Name some detection methods for apoptosis?
By staining with the dye PI (cells fixed)
Phosphatidyl serine, can be detected by incubating the cells with fluorescein-labeled Annexin V, and PI (cells not fixed)
By staining with 7-aminoactinomycin D (cells not fixed)
On a graph, you would see a sub G0 peak showing apoptosis.
State some facts of the fluorochrome 7-Aminoactinomycin D (7-AAD).
Ex: ~488 nm
Em: ~660 nm
DNA-specific
intercalates in G-C regions
long emission wavelength
with FITC & PE labeled Ab for simultaneous evaluation of DNA content and 2-color immunofluorescence using only 488 nm Ex
