FLOW CYTOMETRIC ANALYSIS FLOW CYTOMETRIC ANALYSIS OF THE CELL CYCLEOF THE CELL CYCLE

Jan Bartoš

Laboratory of Molecular Cytogenetics and CytometryInstitute of Experimental Botany

Olomouc, Czech Republic

Introduction to Cell CycleIntroduction to Cell Cycle

CELL CYCLE

• periodic process

• during cell cycle one cell divides into two daughter cells (new

cells can arise only from existing cells during the

cell cycle)

• essential for all organisms (unicellular and multicellular)

• it is precisely regulated

• lost of its control could leads to uncontrolled multiplication of

cells and cancer

Eukaryotic Cell CycleEukaryotic Cell Cycle

consist of four phases:

G1 – preparation of DNA synthesis

S – reduplication of chromosomes

G2 – preparation of mitosis

M – division of nucleus and cells

Daughtercells

Chromatids

Chromosomesegregation and

cell division

M

G 1

G 0

S

G 2

DetectorLight source

PRINCIPLE OF FLOW CYTOMETRYPRINCIPLE OF FLOW CYTOMETRY

Flow cytometry involves the analysis of fluorescence and light scatter properties of particles in flow, moving with respect to the point of measurement

Cell Cycle AnalysisCell Cycle AnalysisCell Cycle AnalysisCell Cycle Analysis

• One of the earliest applications of flow cyto-metry was the analysis of cell cycle position by measurement of cellular DNA.

• Flow cytometry is still the method of choice for fast, accurate determination of cell cycle distributions.

• Recently multiparametric methods were developed, which allow more detailed ana-lysis of the cell cycle.

DNA content during the cell cycleDNA content during the cell cycle

50 0

30 0

40 0

20 0

10 0

00 20 0 40 0 60 0 80 0 10 00

N u c lear D N A co n ten t (ch a n n e l n u m b er)N

umbe

r of

nuc

lei G 1 (2 C )

S

G 2 (4 C )

4 C

2 C

G 1 S G 2 M G 1

DN

A C

onte

nt

C e ll cy cle p h a se

(a) Nuclear DNA content doubles from the 2C level to the 4C level during the S phase of the cell cycle. DNA content returns to the 2C level during mitosis (M), when two daughter nuclei are formed. (b) theoretical histogram of nuclear DNA content.

a b

Histogram of relative nuclear DNA contentHistogram of relative nuclear DNA content

• easy, fast and non-expensive

• good for determination of cell cycle distribution

• cannot study cell cycle kinetics

G1 or S ? S or G2 ?

Detection of DNA synthesisDetection of DNA synthesisDetection of DNA synthesisDetection of DNA synthesis

• A brief pulse of 5-bromo-2’-deoxyuridine (BrdU) can be used for the detection of cells in S-phase.

• BrdU is incorporated into newly synthesized DNA in place of thymidine.

• The incorporated BrdU can be detected with an antibody, identifying those cells that synthesized DNA during the pulse.

• 1982 – human• 1998 – plant

Detection of incorporated BrDetection of incorporated BrdUdUDetection of incorporated BrDetection of incorporated BrdUdU

1

2

3

1) Partial denaturation of DNA (heat, acid, enzyme-DNase I)

2) Immunocytochemical detection of incorporated BrdU (1 step or 2 step procedure)

3) Staining of DNA with fluorescent dye (e.g. PI or DAPI)

4) Flow cytometric analysis

00 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0

R e l a t i v e n u c l e a r D N A c o n t e n t

Brd

U f

luor

esce

nce

1 0 1

10 2

10 3

10 4

Detection of S phase (DNA synthesis) Detection of S phase (DNA synthesis) using flow cytometryusing flow cytometry

G1G2

early S late S• allow kinetic cell cycle

study

• allow better determination of cell cycle distribution

• time consuming

0 200 400 600 800

DNA content [r.u.]

Brd

U f

luo

res

ce

nc

e [

r.u

.]

103

101

100

104

a

0 200 400 600 800 1000

DNA content [r.u.]

Brd

U f

luo

res

ce

nc

e [

r.u

.]

103

101

100

104

b

0 200 400 600 800 1000

DNA content [r.u.]

Brd

U f

luo

res

ce

nc

e [

r.u

.]

103

101

100

104

c

DNA/BrdU analysisanalysis of the cell cycle

Cell cycle analysis in Vicia faba root tips. Root tips were incubated with BrdU for 1 hour.Incorporated BrdU was detected via indirect immunofluorescence: imme-diately after the BrdU pulse (a); 1 hour after the pulse (b) and 4 hours after the pulse (c).

c.TG2+M = ln[1+f uG2+M(t)] + c.t 0 < t < TG2+M

c.(TS+TG2+M) = ln[1+f lu(t)] + c.t TG2+M < t < Ts+TG2+M

c.TG2+M = ln[1 – f ld(t)/2] + c.t TG2+M < t < Ts+TG2+M

Tc = ln(2p)/c

White et al. 1990

f uG2+M

fraction of unlabeled undivided cells

f lu fraction of labeled undivided cells

f ld fraction of labeled divided cells

p fraction of cycling cells

Calculation of cell cycle parametersCalculation of cell cycle parameters

R ela tiv e n u c lear D N A co n ten t

Brd

D f

luor

esce

nce

00 20 0 40 0 60 0 80 0 10 00

10 1

10 2

10 3

10 4

00 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0

R e l a t i v e n u c l e a r D N A c o n t e n t

Brd

U f

luor

esce

nce

1 0 1

10 2

10 3

10 4

f uG2+M

fraction of unlabeled undivided cells

f lu fraction of labeled undivided cells

f ld fraction of labeled divided cells

p fraction of cycling cells

f uG2+M

f lu f lu

f ld

Distribution of labelled population (BrdU positive) immediately (a) and 6 hours (b) after the BrdU pulse. Note definition of individual fractions.

a b

Regulation of Regulation of eukaryoticeukaryotic cell cycle cell cycle

Leland Hartwell, Tim Hunt and Paul Nurse - Nobel Prize in 2001 for their discoveries of “key regulators of the cell cycle”.

mitotic cyclin

Mitotic CDK

degradation ofmitotic cyclin

S-phase cyclin

S-phase CDK

degradation ofS-phase cyclin

active MPF – enter of mitosis

active S-phase CDK – start of DNA replication

Proteins involved in the cell cycle control:• cyclins•CDK – cyclin dependent kinases• CKI – cyclin dependent kinase inhibitors• phosphatases • other proteins (pRB – retinobastoma proteins, APC, transkription factors, etc.)

Cell cycle is regulated by both extra-cellular and intracellular signals.

Green Fluorescent Protein (GFP)Green Fluorescent Protein (GFP)

• GFP is naturally occurring protein

from the jellyfish Aquorea victoria. • Wild type GFP fluorescence around

510 nm after excitation with UV or

488 nm laser.• Many variants of GFP were

developed (including BFP, YFP,

EGFP,…)• GFP can be used as reporter gene.• GFP remain fluorescent after fusion

with another protein.

Fluorescence of GFP fused with different variants of p34cdc2 in nuclei isolated from tobacco: wild type p34cdc2 (a); mutants of p34cdc2 (b,c); control - nuclei without GFP.

Analysis of p34Analysis of p34cdc2cdc2 expression in tobacco plant expression in tobacco plant

1 10 100 1000 10000

GFP fluorescence [r.u.]

nu

mb

er

of

ev

en

ts

control

cdc2-GFP

a

1 10 100 1000 10000

GFP fluorescence [r.u.]

nu

mb

er

of

ev

en

ts

controlcdc2-161-GFP

b

1 10 100 1000 10000

GFP fluorescence [r.u.]

nu

mb

er

of

ev

en

ts

control

cdc2-14-GFP

c

THE LABORATORYTHE LABORATORY

http://www.ueb.cas.cz/olomouc1