Flow Cytometry: Introduction

Question 1

What is flow cytometry?

Answer 1

A technique that simultaneously measures several physical characteristics of a cell in suspension. This is done by light scatter and fluorescence.

Question 2

What is Fluorescence-Activated Cell Sorting (FACS)?

Answer 2

Sorting (separating) cells based on properties measured in flow

Question 3

What can a flow cytometer tell us about a cell?

Answer 3

1. Relative size
2. Relative granularity
3. Relative fluorescence intensity

Question 4

Describe the key differences between flow cytometry and fluorescence microscopy.

Answer 4

• Microscopy can only visualise small numbers of cells (in a single field) at a time compared to flow cytometry.
• Flow cytometry can compare characteristics of multiple cell populations. This would be much harder to do with microscopy.
• In microscopy quantitation of fluorescence intensity is subjective and therefore imprecise compared to flow cytometry.

Question 5

Describe the 3 basic components of a flow cytometer.

Answer 5

1. Fluidics - cells in suspension flow in single-file
2. Optics - illuminated cells scatter light and emit fluorescence
3. Electronics - data is collected, filtered and converted to digital values which are stored on the computer

Question 6

How is a single-file suspension of cells achieved?

Answer 6

• Inject cells into a sheath fluid as it passes through a narrow orifice (50-300uM)
• 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 - ensures cells flow in single-file

Question 7

What does light scatter tell us about size and granularity?

Answer 7

• Forward light scatter is proportional to size
• 90 degree light scatter is proportional to granularity
• We can measure size and granularity without fluorescence

Question 8

What does a dot plot show?

Answer 8

• Different populations of cells cluster together based on size and granularity
• Cells appear as dots on graph
• Can identify different cell populations and their characteristics

Question 9

Why does flow cytometry require filters and mirrors?

Answer 9

• The 4 different colours of light used have overlapping emission spectra
• Diverting light enables us to cut-out overlap and only collect light from the specific fluorochrome that is desired
• The 4 colours can then be quantified separately

Question 10

What do the electronics do in the flow cytometer?

Answer 10

Change light into digital information - analog-digital conversion

Question 11

Label a diagram of a flow cytometer.

Answer 11

Question 12

Label a diagram of the channel layout in flow cytometry.

Answer 12

Question 13

What is Stokes shift?

Answer 13

The energy difference between the lowest energy peak of absorbance and the highest energy of emission, measured in wavelength (nm) or frequency (hz) units.

Question 14

Name 3 fluorochromes - immunofluorescent dye molecules.

Answer 14

1. Fluorescein isothiocyanate (FITC) - green
2. Phycoerythrin (PE) - orange
3. Peridinin Chlorophyll Protein (PerCP) - red

Question 15

Explain how and why combinations of fluorochromes are used together.

Answer 15

• The more fluorochromes expressed, the greater the number of cell populations possible.
• Gating can then be used to visualise different cell populations separately.
• Using combinations of fluorochromes allows us to compare the characteristics of more cell populations at the same time.

Question 16

Describe the direct and indirect methods of fluorescent labelling.

Answer 16

• Direct - Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes
• Indirect - unconjugated MoAbs added and then a secondary preconjugated antibody is added

Question 17

What are the advantages and disadvantages of the indirect method of fluorochrome labelling?

Answer 17

Advantages:

• Signal can be amplified as more than one secondary MoAb can bind to the primary MoAb

Disadvantages:

*

Question 18

What are the 2 classic ways of displaying fluorescence data?

Answer 18

• Histogram - all we can quantitate is the number of cells positive for the fluorochrome
• 2D dot-plot - can look at 2 parameters and multiple populations at the same time - size and granularity