Fluorescent light microscopy showing mitosis, Fluorescent light microscopy showing mitosis, especially immunolabelled cytoskeleton and tubulinespecially immunolabelled cytoskeleton and tubulin

CELL REPLICATIONCELL REPLICATION

chromosomes

tubulins

actin

centriole

Cell REPLICATIONPROLIFERATIONMUTIPLICATIONDIVISION

G0resting

Fluorescent light microscopy showing mitosis, Fluorescent light microscopy showing mitosis, especially immunolabelled cytoskeleton and tubulinespecially immunolabelled cytoskeleton and tubulin

CELL REPLICATIONCELL REPLICATION

chromosomes

tubulins

actin

centriole

EM showing mitosis in a satellite cellEM showing mitosis in a satellite cell

G0resting

main METHODS

1. Incorporation of EXOGENOUS label into newly synthesised DNA

(a) Radioactive tritiated thymidine (3HTdr) : visualise by autoradiography or quantitate by scintillation

(b) Thymidine analogue, 5 bromode-oxyuridine (BrdU): detect with antibody

2. Detection of ENDOGENOUS protein expressed only in replicating cells

main METHODS

1. Incorporation of EXOGENOUS label into newly synthesised DNA

(a) Radioactive tritiated thymidine (3HTdr) : visualise by autoradiography or quantitate by scintillation

(b) Thymidine analogue, 5 bromode-oxyuridine (BrdU): detect with antibody

2. Detection of ENDOGENOUS protein expressed only in replicating cells

• Picture of gut

Synthesis of new DNALabel T with 3HTdr or BrdU

(1a) Tritiated thymidineExogenous radioactive label

incorporated during DNA synthesis

Detection by autoradiographyshows LOCATION of labelled nuclei/cells

• Cut fixed tissue sections onto slides.• Dip slides into photographic emulsion. • Leave for weeks in the dark. • Decay of isotope affects emulsion ‘virtual image’.• Develop film: causes silver grains to deposit.• Observe silver grains in emulsion (above tissue)

Sampled at 1 hour after labelling= pre-mitotic nuclei labelled

Sampled at 2 weeks after labelling= post-mitotic muscle nuclei labelled

Tissues sampled at 2 weeks after label= labelled post-mitotic muscle nuclei

CRUSH INJURY

Grounds & McGeachie, 1989

Compare CRUSH INJURYwith events afterMUSCLE GRAFTING

McGeachie & Grounds, 1990

McGeachie & Grounds, 1989

Tritiated thymidineExogenous radioactive label incorporated

during DNA synthesis Advantages1. persistent label in new DNA 2. can track fate of labelled cells – where it moves. 3. labels daughter cells and can track4. can calculate number of divisions.

Disadvantages1. radioisotopes2. need to administer the label to cells or animals3. dilution of the label/nucleus with each cell

division4. possible re-utilisation of label (if cell dies and is

phagocytosed by macrophages)

Tritiated thymidineExogenous radioactive label

incorporated during DNA synthesis

• QUANTITATION by scintillation counting

Measures TOTAL label present

α- and β- tubulin

Quantitation of cell replication: Scintillation counterIn tissues or cells in culturee.g 96 well plateAllows comparison of many culture conditions. Samples often in triplicate.

METHOD (to QUANTITATE cell proliferation)• Cells in culture (96 well plate) grown +/- growth factors for various times.• Before sampling, wells are exposed to tritiated thymidine for 4 hrs.• Radioactivity is incorporated into new DNA (nuclei in S phase) of cells• Excess tritiated thymidine is washed off.

•Radioactivity of ALL cells/well is measured in a scintillation counter•Total radioactivity measures the number of cells/myoblasts in S phase.•Compare mitogenic effects of different growth factors

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1.0

1.5

2.0

2.5

3.0

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1.0

1.5

2.0

2.5

3.0

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1.0

1.5

2.0

2.5

3.0

0 0.01 0.1 1 10

0 0.01 0.1 1 10 0 0.01 0.1 1 10

0 0.01 0.1 1 10 0 0.01 0.1 1 10

bFGF

TGF-1

SJL/JBALB/c

Fo

ld In

crea

se in

3H

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ine

Up

take

Effect of Growth Factors on Myoblast Proliferation

METHOD (to QUANTITATE cell proliferation)• Compare cells from 2 strains of mice• In response to different concentrations of GF in vitro• Compare 2 GF (FGF and PDGF)

•SHOWS that FGF has greater mitogenic effect.•PDGF has little effect at low conc. and has less effect on proliferation

Growth factor (ng/ml)

PDGFPDGF

EXERCISE: Cell Replication

You have labelled a population of replicating cells in the developing brain of a newborn mouse by systemic injection of tritiated thymidine.

You sample several brains from the young animals within one day of injection to determine the initial level of labelling.

One year later you sample the other brains. With respect to the initial population of labelled cells you want to know:• if they divided many times after birth • where they moved to in the brain • if any of them were lost from the tissue (died or exited).

With respect to the above 3 points:1. What would examination of tissue sections tell you? 2. What would extraction of the total DNA and scintillation counting tell

you? 3. If the labelled cells had divided many times during the year (but none

had been lost) what would you see on tissue sections and what would you see on scintillation counting?

  

(1b) Bromodeoxyuridine (BrdU)

BrdU is an analogue of thymidine. Exogenous BrdU is incorporated during

DNA synthesis

BrdU is detected by an ANTIBODY

Primary muscle cell cultureBrdU = red (DNA synthesis in nucleus)Desmin = green (identifies myoblasts)

BrdUExogenous label incorporated during

DNA synthesis Advantages1- 4. as for tritiated thymidine

5. Antibody detection is very quick and not hazardous.

Disadvantages2- 4. as for tritiated thymidine.

BrdU can be toxic and interfere with cell biology . Therefore usually used for short–term studies i.e label and sample, especially in tissue culture.

(2) ENDOGENOUS proteins that change during the cell cycle

e.g proliferating cell nuclear antigen (PCNA)

No need to add anything to label the cells:

Just DETECT what is already there

Use an antibody to detect

PCNA immunostaining (brown) identifies replicating cells in damaged skeletal muscle tissue (TS). Double staining needed to identify cell type: many of these may be macrophages..

Immunostaining to detect proteins involved in cell proliferation (tissue sections or any cells):

- PCNA (or Ki67)COUNT labelled nuclei to measure numbers of proliferating cells (Can double stain to identify specific cell e.g myoblasts)

Tissue Culture Immunostaining to detect proteins involved in cell proliferation

-PCNA-Ki67

(2) ENDOGENOUS proteins that change during the cell cycle e.g PCNA

Advantage1. No need to add anything to label the cells2. Detect by antibody

Disadvantage1. Detects cells while they are replicating only.

Therefore no good for tracking cell FATE.

(3) Cell assays to measure NUMBER of cells,

by metabolites produced

e.g “CellTiter” assay

No need to add anything to label the cells:Just DETECT what is already there

Cell Metabolites

• The CellTiter 96® assay is a colorimetric method that determines the number of viable cells in a culture.

• A component of the assay system, Owen’s reagent, is reduced by living cells to a soluble coloured formazan product.

• Colour development is monitored by recording the absorbance at 490nm.

• The extent of colour development directly relates to the amount of formazan product which is in turn directly proportional to the number of living cells.

• Measures NUMBER. This is balance of cell proliferation AND cell death: need to consider. e.g could have high division + high death to result in little change,

or high division + no death = BIG increase