© dr m.a. hill, 2006 - slide 1 anat3231 - cell biology laboratory 2 school of medical sciences the...

© Dr M.A. Hill, 2006 - slide 1

ANAT3231 - Cell BiologyLaboratory 2

School of Medical SciencesThe University of New South Wales

Dr Mark HillCell Biology Laboratory

Room G20 Wallace Wurth Building

Email: [email protected]

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UNSW Copyright Notice

© Dr M. A. Hill, 2006Cell Biology Laboratory

School of Medical Sciences, Faculty of MedicineThe University of New South Wales, Sydney, Australia

COMMONWEALTH OF AUSTRALIACopyright Regulations 1969

WARNINGThis material has been copied and communicated to youby or on behalf of the University of New South Wales

pursuant to Part VB of the Copyright Act 1968 (the Act).The material in this communication may be subject to

copyright under the Act. Any further copying orcommunication of this material by you may be the subject

of copyright protection under the Act.Do not remove this notice.

Web: http://anatomy.med.unsw.edu.au/cbl/cbl.htmEmail: [email protected]


Tissue Culture References

• Visit the CBL Tissue Culture Lab• http://anatomy.med.unsw.edu.au/cbl/lab/visit_tc.htm

– Note this is an old page that may soon be removed.

• UNSW Cell Biology - Lab Methods– http://cellbiology.med.unsw.edu.au/units/lab/cblmethod_tc01.htm




Cell Analysis





+ Growth Factor


Lifespan Processes

• Birth– Mitosis

• Except germ cells– meiosis

• Death– Apoptosis

• Programmed cell death

– Necrosis

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Image source- http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/intro.html


Cell Cycle- External Regulators• External factors

– can also regulate progression through cycle

• Cell replacement in different tissues– regulated by polypeptide

growth factors• factor can be specific for

specific cell types

Image: MBoC Ch18 Fig 44

© Dr M.A. Hill, 2006 - slide 8 Image: MBoC Ch18 Fig 45

Growth Factor Model• Fibroblasts in culture• Serum- Proliferation (prepared by clotting)• Plasma- No proliferation (Prepared by centrifugation)• Clotting

– Allows platelets to release secretory granules– Platelet-derived growth factor (PDGF)

• CT cells express receptors bind small PDGF glycoprotein


Retinoblastoma Protein (Rb)

Image: MBoC Ch18 Fig 46/47

Image: Cell Ch15Fig33




Sterile Stocks

• Before anything is done in Lab– hands are washed– PPE gloves and gown are worn

• All bottles, instruments, gowns etc are sterilized in bags, and then stored in a glass front cupboard

• Everything to be used is opened just prior to use washed with ethanol and then placed in sterile hood


Sterile Solutions• made and stored in different ways

– depend on stability of components within solution– solutions bought commercially already sterile either frozen

or as a liquid• these are generally filter sterilized

• Media– can be prepared from premixed powder with sterile water

followed by pH adjust and filtering• Solutions can be made from their raw ingredients

– usually salts, buffer, pH indicators, amino acids, essential compounds– Mixed, pH adjusted and filtered– Filters are membranes with a 0.2 micron pore (hole) size– Water is of very high purity

» filtered and/or autoclaved




Frozen Cells• Cells are stored frozen in

liquid nitrogen (-180C)– storage tanks contain small

vials of the cells in a freezing solution

• that prevents ice crystals forming and damaging the cells

– vials are held in long canes• of 5-6 vials• stored in 6 individual cannisters• several hundred vials can be

stored in a single vessel• cells can at a later time be

thawed and begin to grow again

– Cell suppliers






Solutions and Plates• Sterile solutions and tissue culture

plates coated with adhesive molecules– stored in fridge at 4C or freezer -20C

• Tissue culture plates and other items are warmed to room temperature– while most solutions are warmed to

37C in a water bath just before use

• Everything is then placed in the sterile hood




Sterile hood and incubator• Cell work requires a sterile work area

– use a Biohazard Class 2 Lamina Flow Hood• filters air and passes it over an enclosed work surface• filtered barrier of air also protects the researcher




Cell Incubator

• incubator maintains optimal conditions for cell growth– warm (37C)– gas regulated

• 5 - 10% carbon dioxide

– dark, moist environment

• Cells are grown – in special growth solutions

• media

– inside sterile plastic dishes of various sizes and shapes

red colour is from a dye (phenol red) which changes colour dependent upon the pH of the solution




Cell Analysis• Cells growing in dishes can be removed from the

incubator and looked at under a microscope• Cells can be checked

– if they are growing– if any contamination has occurred

• Photos of the cells can be taken and analysed– currently done using a video camera– Computer analysis

• NIH Image




Cell Analysis• Cells can be analysed in many different ways

– Living or Fixed• Flow cytometry• Time lapse• Confocal microscopy• Immunohistochemistry• In Situ hybridization• Biochemically

– DNA (Southern)– mRNA (Northern)– Protein (Western)

• Microarray

© dr m.a. hill, 2006 - slide 1 anat3231 - cell biology laboratory 2 school of medical sciences the...

Documents

cell ch15fig33 slide

copyright act

unsw copyright notice

small pdgf glycoprotein

cell analysis growth

specific cell types

cycle cell replacement

mboc ch18 fig