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Flow cytometry. Powerful technique for analysis of multiple parameters of individual cells within heterogeneous populations multiple physical characteristics of single particles or cells. What is flow cytometry?. scheme of flow cytometry. The fluidic system & optics system.

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Flow cytometry

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Flow cytometry l.jpg

Flow cytometry


What is flow cytometry l.jpg

Powerful technique for

analysis of multiple parameters of individual cells within heterogeneous populations

multiple physical characteristics of single particles or cells

What is flow cytometry?


Scheme of flow cytometry l.jpg

scheme of flow cytometry


The fluidic system optics system l.jpg

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


The fluidic system l.jpg

The fluidic system

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

Laser

Hydrodynamic focusing


Size comparison by flow cytometry l.jpg

Size comparison by flow cytometry

  • ~ 1 ~ 15 uM size detection


Forward scatter l.jpg

Forward scatter

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

  • Cell size


Side scatter l.jpg

Side scatter

  • Granularity and structural complexity inside the cell


Scatter plot l.jpg

Scatter plot

SSC

FSC


Energy state diagram l.jpg

Energy state diagram


Using fluorescence in flow cytometry l.jpg

Using fluorescence in flow cytometry


Fluorescence detection l.jpg

Fluorescence detection


Two color experiment l.jpg

Two color experiment


Two color experiment14 l.jpg

Two color experiment

-/+

+/+

-/-

+/-


Compensation l.jpg

Compensation


Compensation16 l.jpg

Compensation

  • No-stained sample

  • Only FITC stained sample

  • Only PE stained sample

uncompensated

compensated

PE

FITC


Application apoptosis l.jpg

Application (apoptosis)

  • Apoptosis (FSC/SSC)

Apoptosis

SSC

Necrosis

FSC


Application apoptosis18 l.jpg

Application (apoptosis)

  • Apoptosis (Annexin V/PI assay)

Normal Cell

Apoptotic Cell

: Phosphatidyl Serine

: Annexin V-FITC


Application apoptosis19 l.jpg

Application (apoptosis)

  • Apoptosis (Annexin V/PI assay)

Control

Anti-Fas Ab

Propidium Iodide (PI)

Annexin V-FITC


Application cell cycle l.jpg

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


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