7. Flow Cytometry I Intro

Question 1

What is flow cytometry?

Answer 1

• Technique which simultaneously measures several physical characteristics belonging to
a SINGLE CELL in SUSPENSION (measures properties of cells in flow)

• This is done by LIGHT SCATTER and FLUORESCENCE

Question 2

Define flow sorting

Answer 2

• 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

• You can find out about the cell adhesions, receptors and intracellular components, like the enzymes and the cytokines – using fluorescence

Question 4

What are the different methods of visualisation?

Answer 4

• Fluorescence Microsocopy

- Flow Cytometry

Question 5

What are the basics of flow cytomertry?

Answer 5

• FLUIDICS
- cells in suspension
- flow in single-file through
• OPTICS
- an illuminated volume
- scatter light and emit fluorescence
• ELECTRONICS
- Analogue signals (light signals) are collected, filtered and
- converted to digital values
- that are stored on a computer

Question 6

Describe the different places information flows in a flow cytometry.

Answer 6

Light source -> flow chamber -> optical system -> light detectors -> computer

Question 7

Expand on fluidics.

Answer 7

• 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 8

Expand on the light sources that are used.

Answer 8

• 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)

Question 9

Expand on light scatter.

Answer 9

• When the light hits the cell, the light is scattered in 2 directions
• Forward and 90 degree angle.
  
  • Forwards is proportional to the size
  • 90 degrees is proportional to the granularity
• This gives us information about the cell without any fluorescence involved.
• A graph is plotted with the x-axis showing forward scatter and the y-axis showing the 90 degree scatter. - the different cell types accumulate in a certain area ~ so you can quantitate sub-populations of peripheral blood based on their size and granularity

Question 10

What happens when cells are labelled with fluorescence and they are passed through a flow cytometry?

Answer 10

• When the cells are labelled with fluoresce, they do NOT have a forward and perpendicular scatter, but they emit fluoresce
• This shows that the light goes through filters an mirrors and is then detected.

Question 11

Expand on electronics.

Answer 11

• Processing of signals from detectors ~ analog-digital conversion

Question 12

What is a stokes shift?

Answer 12

• The energy difference between the lowest energy peak of absorbance and the highest energy of emission
• When fluorochrome returns to its unexcited state, it emits light at a longer wavelength

Question 13

Examples of flurochromes and dyes

Answer 13

• Fluorescein isothiocyanate (FITC) = GREEN
• Phycoerythrin (PE) = ORANGE
• Peridinin Chlorophyll Protein (PerCP) = RED

Question 14

What happens when there is overlapping between the different colours? (e.g. cells labelled with 4 different colours)

Answer 14

• 4 different colours - labelling the cells with 4 different antibodies, with 4 different colours on them with one laser line.
• The cell emits depending on whether it is positive or negative for a specific antibody
• The light goes through different filters and mirrors before reaching the detector ~ detector recognises a narrow wavelength of light
• Many fluorochromes with different excitation emissions – so you have to choose carefully when you are combining different fluorochromes.

Question 15

Give examples of single cells in suspension

Answer 15

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

Question 16

Describe the different methods of labelling.

Answer 16

• DIRECT: Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes
• INDIRECT: Unconjugated MoAbs ~ you would have your primary specific monoclonal antibody and then in the second step you would have a secondary antibody to the antibody used in the first step (a fluorophore is attached to the secondary antibody)

Question 17

What are the different methods to display data?

Answer 17

• Histogram - Fluoroescence intensity on the X-axis and cell count on the y-axis ~ you can only measure how many cells are fluorescent for that marker
• Dot plot – you have forward scatter on the x-axis and side scatter on the y-axis ~ you can quantitate cells based on 2 parameters

Question 18

Advantage of a dot plot.

Answer 18

You can quantitate 4 populations of cells.

E.g. single positive PE pop, double positive FITC and PE pop, negative pop, and single positive FITC pop.

Question 19

What is gating?

Answer 19

• Gating is where you draw a gate around a specific cell type (on a dot plot) ~ so you can look at the cells in this gate on the basis of their fluroesence.
• histogram analysis of this gate shows 2 populations. - You can then find out what proportion fall into a specific area? and can also quantitate the 4 proportions

Question 20

How many populations do you get with 2 and 3 antibodies?

Answer 20

• With 2, you get 4 populations

- With 3, you get 8 populations