Flow cytometry: introduction and application

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

→ 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)

Question 2

What can a flow Cytometer tell us about a cell?

Answer 2

Relative Size
Relative Granularity/Internal Complexity
Relative Fluorescence Intensity

Question 3

As well as flow cytometry as a method of visualisation, what other method can be used and how does it differ from flow cytometry?

Answer 3

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

Question 4

What are the basics of flow cytometry?

Answer 4

Can be split into 3:
1. Fluidics
cells in suspension flowing in single file

2. Optics
   hit by a laser and scatter light and filtered
3. 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

→ 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

Question 5

What happens in the first stage of Flow Cytometry - Fluidics?

Answer 5

→ 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

→ Laminar flow - Sample fluid flows in a central core that does not mix with the sheath fluid
→Hydrodynamic Focusing - Introduction of a large volume into a small volume

Question 6

How do the cells interact with a laser in the optics stage of flow cytometry?

Answer 6

→ Lasers Single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
→ 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
→ The 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.

Question 7

When does fluorescence happen?
What is Stokes shift?

Answer 7

Fluorescence 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 from lecture for most common one)

Stokes shift is the energy difference between the lowest energy peak of absorbance and the highest energy of emission

Question 7

Name some examples of fluorochromes and their dyes (colours)

Answer 7

most common: all excited at 488nm

Fluorescein isothiocyanate (FITC)
GREEN
emits at around 520 nm

Phycoerythrin (PE)
ORANGE
emits further out at about 580-in the orange zone

Peridinin Chlorophyll Protein (PerCP)
RED
emits even further out at 620-in the red zone

Question 8

In flow cytometry, you need cells in suspension, what are some examples of some ideal cell types?

Answer 8

→ Peripheral blood
→ Bone marrow
→ Fine Needle Aspirate
→ CSF and other fluids
→ Fresh Tissue

Question 9

What are the 2 methods of labelling cells with monoclonal antibodies and fluorochromes?

Answer 9

DIRECT: Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes
In direct, the fluorochrome is conjugated onto the antibody which binds directly onto the antigen.

INDIRECT: Unconjugated MoAbs
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.

Question 10

What fluorescent dye is most commonly used to detect cellular DNA?

Answer 10

→ 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 permeabilisation of the plasma membrane

Question 11

How does an assay work when using PI to check for if a cell is damaged etc?

Answer 11

→ 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

Question 12

What is apoptosis? What are some characteristics of cells when this occurs?

Answer 12

→ 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 13

Name some detection methods for apoptosis?

Answer 13

→ 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.

Question 14

List some applications of Flow Cytometry

Answer 14

→ 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

You can use it to purify for example: you add dye to cells and then purify then monitor and see if it has been purified