Fluorescent light microscopy showing mitosis, especially immunolabelled cytoskeleton and tubulin CELL REPLICATION chromosomes tubulins actin centriole

Cell REPLICATION PROLIFERATION MUTIPLICATION DIVISION

G0 resting

Fluorescent light microscopy showing mitosis, especially immunolabelled cytoskeleton and tubulin CELL REPLICATION chromosomes tubulins actin centriole

EM showing mitosis in a satellite cell

G0 resting

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 DNA Label T with 3HTdr or BrdU

(1a) Tritiated thymidine Exogenous radioactive label incorporated during DNA synthesis Detection by autoradiography shows 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 INJURY with events after MUSCLE GRAFTING McGeachie & Grounds, 1990

McGeachie & Grounds, 1989

Tritiated thymidine Exogenous radioactive label incorporated during DNA synthesis Advantages 1. persistent label in new DNA 2. can track fate of labelled cells – where it moves. 3. labels daughter cells and can track 4. can calculate number of divisions. Disadvantages 1.radioisotopes 2.need to administer the label to cells or animals 3.dilution of the label/nucleus with each cell division 4. possible re-utilisation of label (if cell dies and is phagocytosed by macrophages)

Tritiated thymidine Exogenous radioactive label incorporated during DNA synthesis QUANTITATION by scintillation counting Measures TOTAL label present

α- and β- tubulin

Quantitation of cell replication: Scintillation counter In tissues or cells in culture e.g 96 well plate Allows 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

bFGF TGF-  1 SJL/J BALB/c Fold Increase in 3 H-thymidine Uptake 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) PDGF

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 culture BrdU = red (DNA synthesis in nucleus) Desmin = green (identifies myoblasts)

BrdU Exogenous label incorporated during DNA synthesis Advantages as for tritiated thymidine 5. Antibody detection is very quick and not hazardous. Disadvantages 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 Advantage 1.No need to add anything to label the cells 2.Detect by antibody Disadvantage 1.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