By Elzbieta Petelenz

2007/09/17

Flow cytometry
a very general introduction

Principle:
• cells pass in a narrow stream through a laser beam
• their light-absorbing or fluorescing properties are measured
• cells can be separated based on the properties measured in the flow
therefore: the method allows distinguishing cell fractions within a population

Flow

Most commonly used measured characteristics:

• forward scatter (FSC, FALS) – correlates with the size of the cells, provided that the
cells have a bigger diameter than that of the analysing beam
• side scatter (SSC) – reflects the granularity of the cells (no explicit measure of
anything)
• fluorescence – if a fluorescent dye is introduced into the cells (by staining with a dye
or expression of a fluorescent protein), the fluorescence can also be measured

Fluorescence
signals

Focused laser 1
beam
 By Elzbieta Petelenz
2007/09/17

Forward Angl

Laser

90 Degree L

Laser

By Dr. J. Paul Robinson, Purdue University Cytom

2
 By Elzbieta Petelenz
2007/09/17

Fluorescen

Freq
Laser

Fluores
(PMT
By Dr. J. Paul Robinson, Purdue University Cytomet

3
 By Elzbieta Petelenz
2007/09/17

Instrument: BD FACS Aria

A flow cytometer consists of 3 main modules:
• fluidics – the cells are hydro dynamically focused by a sheath fluid (consists mostly of
PBS); they pass through a small orifice (nozzle, 70-100 µm width, analysed cells
should not be bigger than 25-30% of nozzle diameter) which forms droplets,
containing (preferably) individual cells
• optics – the cell-containing droplets pass in front of an analysing laser beam; the
scattered/emitted light is collected by a detector
• electronics – analogue information from the detector (light intensities) are converted to
digital signals, which are recorded by the software

Pulse generation

Time of flight (=pulse width)

4
 By Elzbieta Petelenz
2007/09/17

The data from FACS can be presented in different ways. The most common ones are
• dot plots
• histograms

Advantages
• Isolation of cell sub-populations
• High throughput
• High speed
Disadvantages
• No cell images visible
• Data interpretation
• Size limit