Lecture 1 - Flow Cytometry

Question 1

What is fluorescence?

Answer 1

Fluorescence is the emission of light by a substance as it returns to ground state subsequent to being excited through the absorption of light

Question 2

What is a fluorochrome?

Answer 2

A molecule that is fluorescent

Question 3

Compare SSC and FSC

Answer 3

SSC: side scatter
• Caused by granularity and internal structural complexity
• Structurally complex cells produce a great amount of SSC and vice versa

FSC: forward scatter
• Encodes information about the size of the cells
• Small cells produce a small amount of FSC and vice versa

Question 4

Describe the important components of flow cytometry

Answer 4

Fluidics
• Transport of particles in a stream to the laser beam for interrogation

Optics
• Lasers that illuminate a particle
• Optical filters that direct the signal to the appropriate detectors

Electronics
• Conversion of detected light signals into electrical signals (voltage pulses)
• These are then processed by a computer

Question 5

What are the different ways that fluorochromes can be used?

Answer 5

• Direct conjugates
• Indirect conjugates (secondary labelling)
• DNA labelling
• Cytoplasmic dyes

Question 6

What is spectral overlap?

Answer 6

Some light from one filter will be detected by a different filter

Need to adjust for this
This is called compensation

Question 7

Compare the size and granularity of the following cells:
 • Neutrophils
 • Lymphocytes
 • Monocytes
 • DCs

Answer 7

Neutrophils:
• Large, granular

Lymphocytes
• Small, low granularity

Monocytes:
• Intermediate size and granularity

DCs:
• Intermediate size and granularity

Question 8

Describe compensation

Answer 8

Always have a background population of cells that are not stained with any Ab

Allows correction of Spectral overlap

To compensate:
Subtract FITC (FL1) from PE(FL2)
i.e. PE (FL2) - FITC (FL1)