L8 - Flow Cytometry (Intro and Application) Flashcards
What is Flow Cytometry and what does it depend on?
A technique that enables us to simultaneously measure several characteristics of a single cell in suspension.
It is done by the cells ability to scatter light and emit fluorescence.
What is Flow Sorting?
Sorting and 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
- Cell surface receptors, inter cellular cytokines, cell cycle, viability and apoptosis can be measured in FC.
What is Flow Microscopy and what are its problems?
Using a microscope and fluorescence to measure size and shape etc of a cell.
- not very quantitative: many fields of the microscope would have to be scanned to be able to quanitate cells accurately.
- Rare cells cannot be quanitated in flow microscopy
- Difficult to tell the brightness of cells and there fluorescence by eye in a microscope (cytometry does this for you)
What are the 3 stages of Flow Cytometry?
1) Fluidics
2) Optics
3) Electronics
What happens in the Fluidics phase of Flow Cytometry?
Cells in suspension flow in a single file.
This is accomplished by injecting sample into a sheath fluid as it passes through a small orifice.
This is Hydrodynamic focusing - when a large volume is introduced into a small volume.
What happens in the Optics phase of Flow Cytometry?
Usually a single wavelength of light (laser line), or rarely a mixture of wavelengths.
Can provide milliwatts/watts of light.
It provides coherent light
when light hits the cell - forward light scatter is emitted which is proportional to the size of the cell
Light is also emitted at a 90 degree angle to the cell - which is proportional to the granularity of cell.
What happens in the Electronics phase of Flow Cytometry?
Photo-multiplier tubes convert the light signals into digital signals (analog-digital conversion)
What is Fluorescence
It is the energy difference between the lowest energy peak of absorbence and the highest energy of emission.
When a laser hits a fluorochrome, it is excited at one wavelength, and when it goes back to its unexcited state, it emits fluorescence at a higher wavelength.
State 3 common Fluorochromes and dyes and their colours?
Fluoroescein isothiocyanate (FITC) - green
Phycoerythrin (PE) - orange
Peridinin Chlorophyll protein (PerCP) - Red
What are some ideal samples for Flow Cytometry?
peripheral blood bone marrow needle aspirate CSF fresh tissue
What are the 2 ways to label cells in FC?
DIRECT - Monoclonal antibodies (MoAbs) are preconjugated to flourochromes
INDIRECT - Unconjugated MoAbs
What dye is used to detect DNA and why?
Propidium Iodide is usually used.
It undergoes a dramatic increase in fluorescence when binding to DNA
It requires the permeabilisation of plasma membrane
How can Propidium Iodide be used to check cell viability?
PI cannot usually cross an intact cell membrane - if PI penetrated the cell membrane it is assumed to be damaged
Cells that are brightly fluorescent with the PI are damaged or dead
What are 3 methods for detecting Apoptosis
- use of PI
- Phosphatidyl serine would be outside the membrane in apoptosis, so annexin 5 can be used to detect this
- staining with 7AAD
