Flow Cytometry

Anna Merca - Philippe Melas - Tolga Sütlü

Stockholm Research School in Molecular Life Sciences

14 October 2005

Outline

• IntroductionIntroduction

• How Does Flow Cytometry Work?How Does Flow Cytometry Work?

• ApplicationsApplications

• Focus on Analysing ApoptosisFocus on Analysing Apoptosis

• Cell SortingCell Sorting

• Benefits & LimitationsBenefits & Limitations

Introduction

• Powerful analytical tool

• up to 10000 individual cells/sec

• many properties at the same time

• wide area of application

How does it work?

• Cells in suspension (usually stained)

• 5 basic units– Flow cell – Light source– Optical filter units– Photomultiplier tubes– Data processing and operating unit (software)

Direct beam stopLaser

Light

High angle scatter :(Side Scatter)Cell structure

Low angle scatter :(Forward Scatter)Cell size

Fluorescence at longerwavelengths

Flow cell

Direction of flow

Basic Optics of a Flow Cytometer( An automated fluorescent microscope)

Dichroic mirrors 1 2 3

Laser(s)

Cell

CollectionLenses

ScatterLow & High

anglePhotomultiplier tubes

Analysis Applications

• Viability and physiological state• Cell cycle analysis & nucleic acid content• Cell growth & death rates• Intracellular calcium concentration• Apoptosis• Biotechnological Applications

– Bacterial Cultivations– Yeast Cultivations– Mammalian Cell Cultivations

Analyzing the graph-one color-

10 1 10 2 10 3 10 4

CD3 FITC -->

01

0

Num

ber

CD3

NUMBER

10 1 10 2 10 3 10 4

CD3 FITC -->

01

0

Num

ber

CD4

NUMBER

10 1 10 2 10 3 10 4

CD3 FITC -->

01

0

Num

ber

CD8

NUMBER

Analyzing the graph

CD4

10 1 10 2 10 3 10 4

CD8 TC -->

10

11

02

10

31

04

Anti-

TC

R-g

am

ma-d

elta

-1 P

E -

->

10 1 10 2 10 3 10 4

CD3 FITC -->

01

0

Num

ber

0 1

10

21

03

10

4

010

CD3

10 1 10 2 10 3 10 4

CD8 TC -->

10

11

02

10

31

04

Ant

i-TC

R-g

amm

a-de

lta-1

PE

-->

CD3

CD4

CD3+CD4+ green

CD3-CD4+ cyan

CD3+CD4- cyan

CD3-CD4- black

Apoptosis vs. Necrosis

• Suicide

• Genetically programmed

• Cells shrink

• Condensation of chromatin

• Internucleosomal degradation of DNA

• Membrane retains its integrity

• MMP reduced

• Murder

• Random Event

• Cells swell

• Non-specific loss of chromatin structure

• Non-specific degradation of DNA

• Membrane loses its integrity

Apoptosis vs. Necrosis

How to study apoptosis:

Caspases• Fact : Caspase 3 is activated in apoptosis

How to study apoptosis: Mitochondrial Membrane Potential

• Fact: MMP is reduced during apoptosis

How to study apoptosis: Plasma Membrane

• Fact: Phosphatidyl serine (PS) flips from inside to the outside of the membrane during apoptosis

Cell Sorting 1

• sample nozzle is vibrated

• sample stream breaks up into regular droplets

• droplets are electrostatically charged prior to passing through the laser

• droplets containing cells of interest (as characterized by scatter or fluorescence properties) are deflected into tubes by passing them through a pair of charged plates

Cell Sorting 2

ChargedPlates

Collection Tubes

Benefits of Flow Cytometry

• measurement of single cells, identification of sub-populations

• wide area of application for analysis & diagnosis

• efficient and fast

• may be controlled remotely (online)

Limitations

• can not tell the intracellular location and distribution of proteins

• aggregates or debris can give false results• pre-treatment of the cells for fluorescent

staining is time-consuming

• samples such as tissue or cells in culture have to be treated to separate cells

• expensive and needs highly-trained technicians

Thanks for listening...