Flow Cytometry

Question 1

What is flow cytometry?

Answer 1

Technique which simultaneously measures several physical characteristics belonging to a SINGLE CELL in SUSPENSION
This is done by LIGHT SCATTER and FLUORESCENCE

Question 2

What is the difference between flow cytometry and flow sorting?

Answer 2

Flow Cytometry
- Measuring properties of cells in flow
Flow Sorting
- Sorting (separating) cells based on properties measured in flow
- Also called Fluorescence-Activated Cell Sorting (FACS)

Question 3

What can a flow cytometer tell us about a cell?

Answer 3

1. Its Relative Size
   
   2. Its Relative Granularity/Internal Complexity
   3. Its Relative Fluorescence Intensity

Question 4

What are the downsides of using fluorescence microscopy as a method of visualisation?

Answer 4

Fluorescence microscopy-

- not very quantitative as seen below, cannot quantitate rare cells either
- Have to quantitate fluorescence by eye which is generally subjective

Question 5

What are the basic stages of flow cytometry?

Answer 5

Fluidics, Optics and Electronics

- Cells in suspension (f)
- flow in single-file through
- an illuminated volume where they (o)
- scatter light and emit fluorescence
- that is collected, filtered and
- converted to digital values (e)
- that are stored on a computer

Question 6

How is the fluidics portion of flow cytometry achieved?

Answer 6

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

Question 7

What are the light sources used in flow cytometry?

Answer 7

Lasers

- Single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
- can be inexpensive, air-cooled units or expensive, water-cooled units
- provide coherent light (light emitted at a single frequency)

Question 8

What happens when a laser hits a cell in flow cytometry?

Answer 8

Light scatters in a forward direction which is proportional to the size of the cell
- That is called forwards light scatter
Light also scatters at a 90 degrees which is a measure of the granularity of the cell
- Called Side Scatter

Question 9

How can you separate cells by fluorescence?

Answer 9

Each filter has a threshold for how much light can pass through and hit these PMTs
(photomultiplier tubes) which detect fluorescence
Then comes the processing of signals from detectors through analog-digital conversion

Question 10

How does fluorescence work in general? What is Stokes shift?

Answer 10

Fluorescence happen when laser hits the fluorochrome and is excited at one wavelength and then when it goes back to its unexcited state it emits fluorescence at a higher wavelength
Stokes Shift
- Is the energy difference between the lowest energy peak of absorbance and the highest energy of emission

Question 11

What are the different fluorochromes and dyes?

Answer 11

Fluorescein isothiocyanate (FITC) GREEN
Phycoerythrin (PE) ORANGE
Peridinin Chlorophyll Protein (PerCP) RED
There’s always overlap so those filters help get rid of the overlap

Question 12

What are the different methods of labelling in immunofluorescence?

Answer 12

• DIRECT : Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes
  
  • INDIRECT: Unconjugated MoAbs
    So in the indirect we are labelling with the monoclonal antibodies and then coming in with an antibody to the monoclonal that has the fluorochrome attached to it

Question 13

What are the different ways we can display flow cytometry data?

Answer 13

The histogram is a 1 dimensional display so we can only investigate one fluorescence/parameter at a time
In a dot plot we can analyse two parameters at the same time

Question 14

What is gating in flow cytometry?

Answer 14

We can draw a gate around the population we want to have a closer look at so the computer will only measure the fluorescence of cells from that population

For example we can ask the computer to show the cells satisfying gate A on the basis of just FITC fluorescence (one parameter so in a histogram); shows which ones are CD3-FITC positive and negative

Question 15

What are univariate cell cycle methods?

Answer 15

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.

Question 16

How does the PI assay work?

Answer 16

• 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

Question 17

What changes occur during apoptosis that we could measure?

Answer 17

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

Question 18

How can we detect apoptosis?

Answer 18

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)

Question 19

What would cell apoptosis usually look like on a cell count-PI graph?

Answer 19

Sub-G0 peak show us the apoptotic cells
However the method is not totally reliable as it could be DNA fragments and some cells do not show that peak when they apoptose

Question 20

How can we tell the difference between live, early apoptotic and late apoptotic/necrotic cell?

Answer 20

The phosphatidyl serine are usually in the cell
So in a live cell the plasma membrane is intact so annexin wouldn’t bind to phosphatidyl serine and PI will not get in either so it would be negative for both
Early apoptotic cells have phosphatidyl serine on the outside of the cell so annexin can bind but PI still cannot cross the membrane as it is still intact
- So it would be positive for annexin and negative for PI
In late apoptotic/necrotic cells the annexin would be able to bind to the phosphatidyl serine and the membrane is no longer intact so the PI can get inside
- So it would be positive both

Question 21

How does 7-AAD bind to cells?

Answer 21

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

Question 22

How are cell sorted in a flow cytometer?

Answer 22

We have the nozzle tip and the sample entering the nozzle tip and flowing in single file past the laser
Then the cell emitting light which is detected and displayed
The difference is that now as well as providing that analysis we can now draw regions around the cells that we want to isolate and the information feeds back to the sorter
When the machine sees a cell that satisfies the criteria, as soon as the laser hits the screen it tells the machine to wait until that cell gets to the end of the stream
A sort decision is sent to the stream so that when the cell that we want gets to the droplet the machine charges the stream so that the droplet has a charge
This way it can be pulled into a different tube by deflection plates

Question 23

What are the potential applications of flow cytometry?

Answer 23

Immunophenotyping of leukaemias & lymphomas
Detection of MRD
Stem cell enumeration
CD4/CD8 in HIV
Measurement of intracellular cytokines
Study of cell cycle, viability & apoptosis
Measurement of cell proliferation
Assessment of transfection efficiency