Flow Cytometry

Question 1

What is the definition of Flow cytometry

Answer 1

rapid measurement of the optical & fluorescent properties of a large number of particles

Question 2

What particles can be used in Flow Cytometry

Answer 2

Eukaryotic cells
Bacteria
Non-living materials e.g. latex beads

Question 3

What instruments are used in Flow Cytometry

Answer 3

Fluidics system: draws cells into machine & channels into single file.

Optical system: lasers & lenses focus laser beams.

Signal detection & processing system: converts detected light signals to voltages & records this data.

Question 4

How does the fluidics system work

Answer 4

Particles enter flow cytometer through sample injection port.

Fluidics system:
- Central core containing sample fluid.
- Outer sheath fluid:
Fluids interact producing core fluid stream one cell
wide; only one cell can pass through laser beam at a
time = hydrodynamic focussing. (check lecture
image)

Flow rates adjusted:
Slow flow rate = High resolution
Fast flow rate = Low resolution

Samples must be “clean”: free from debris.

Question 5

How does the optical system work

Answer 5

Cell passes through interrogation point = laser beam.

Light hitting a cell divided into:
Forward scatter (FSC): Light in same direction as incident laser beam –> Measures cell size.
Side scatter (SSC): Reflected light approximately 90° to the incident laser beam –> Measures cell granularity.

Emitted light from fluorophores (cells that re-emit light upon light application) registered by corresponding detector.