Flow cytometry

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Flow cytometry
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What is flow cytometry?

• Powerful technique for
• analysis of multiple parameters of individual
  cells within heterogeneous populations
• multiple physical characteristics of single
  particles or cells

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scheme of flow cytometry
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The fluidic system optics system

• heart of the system
• where the laser and the sample intersect and the
  optics collect the resulting scatter and
  fluorescence

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The fluidic system

• Particles or cells are passed through the laser
  beam one at a time

Laser
Hydrodynamic focusing
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Size comparison by flow cytometry

• 1 15 uM size detection

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Forward scatter

• Light is quantified by a detector that converts
  intensity into voltage
• Cell size

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Side scatter

• Granularity and structural complexity inside the
  cell

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Scatter plot
SSC
FSC
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Energy state diagram
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Using fluorescence in flow cytometry
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Fluorescence detection
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Two color experiment
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Two color experiment
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-/
-/-
/-
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Compensation
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Compensation

• No-stained sample
• Only FITC stained sample
• Only PE stained sample

compensated
uncompensated
PE
FITC
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Application (apoptosis)

• Apoptosis (FSC/SSC)

Apoptosis
SSC
Necrosis
FSC
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Application (apoptosis)

• Apoptosis (Annexin V/PI assay)

Normal Cell
Apoptotic Cell
Phosphatidyl Serine
Annexin V-FITC
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Application (apoptosis)

• Apoptosis (Annexin V/PI assay)

Control
Anti-Fas Ab
Propidium Iodide (PI)
Annexin V-FITC
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Application (cell cycle)
Apoptotic DNA Histogram
A typical DNA Histogram
G0-G1
G2-M
S
Apoptotic cells
Normal G0/G1 cells
of Events
Fluorescence Intensity