1. © Dr M.A. Hill, 2006 - slide 1 ANAT3231 - Cell Biology Laboratory 2 School of Medical Sciences The University of New South Wales Dr Mark Hill Cell Biology Laboratory Room G20 Wallace Wurth Building Email: m.hill@unsw.edu.au

2. © Dr M.A. Hill, 2006 - slide 2 UNSW Copyright Notice © Dr M. A. Hill, 2006 Cell Biology Laboratory School of Medical Sciences, Faculty of Medicine The University of New South Wales, Sydney, Australia COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been copied and communicated to you by 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 or communication 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.htm Email: m.hill@unsw.edu.au

3. © Dr M.A. Hill, 2006 - slide 3 UNSW Copyright Notice © Dr M. A. Hill, 2006 Cell Biology Laboratory School of Medical Sciences, Faculty of Medicine The University of New South Wales, Sydney, Australia COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been copied and communicated to you by 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 or communication 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.htm Email: m.hill@unsw.edu.au

4. © Dr M.A. Hill, 2006 - slide 4 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

5. © Dr M.A. Hill, 2006 - slide 5 Cell Analysis + Growth Factor

6. © Dr M.A. Hill, 2006 - slide 6 Lifespan Processes Birth –Mitosis Except germ cells –meiosis Death –Apoptosis Programmed cell death –Necrosis Image source- http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/intro.html

7. © Dr M.A. Hill, 2006 - slide 7 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

8. © 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

9. © Dr M.A. Hill, 2006 - slide 9 Retinoblastoma Protein (Rb) Image: MBoC Ch18 Fig 46/47 Image: Cell Ch15Fig33

10. © Dr M.A. Hill, 2006 - slide 10 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

11. © Dr M.A. Hill, 2006 - slide 11 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

12. © Dr M.A. Hill, 2006 - slide 12 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

13. © Dr M.A. Hill, 2006 - slide 13 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

14. © Dr M.A. Hill, 2006 - slide 14 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

15. © Dr M.A. Hill, 2006 - slide 15 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

16. © Dr M.A. Hill, 2006 - slide 16 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

17. © Dr M.A. Hill, 2006 - slide 17 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