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Introduction to Flow Cytometry Basics

Flow cytometry allows distinguishing cell fractions by passing cells in a narrow stream through a laser beam and measuring their light-absorbing or fluorescing properties (1). Commonly measured characteristics include forward scatter (size), side scatter (granularity), and fluorescence if dyes are used (2). The cells pass through a nozzle into droplets that are analyzed by detectors connected to electronics and software (3). Data is typically presented as dot plots or histograms (4). Advantages include isolation of sub-populations, high throughput, and speed, while disadvantages include lack of images and limits on cell size (5).

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68 views5 pages

Introduction to Flow Cytometry Basics

Flow cytometry allows distinguishing cell fractions by passing cells in a narrow stream through a laser beam and measuring their light-absorbing or fluorescing properties (1). Commonly measured characteristics include forward scatter (size), side scatter (granularity), and fluorescence if dyes are used (2). The cells pass through a nozzle into droplets that are analyzed by detectors connected to electronics and software (3). Data is typically presented as dot plots or histograms (4). Advantages include isolation of sub-populations, high throughput, and speed, while disadvantages include lack of images and limits on cell size (5).

Uploaded by

elanna_o2
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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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

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