Talk:2009 Lab 8

From CellBiology

--Mark Hill 11:21, 8 April 2009 (EST) General links for TC


Sigma

learning-center

Sigma Cell Culture Manual (on line)

Cell Culture Books

ECACC Handbook Cell Culture Virtual Stockroom

GIBCO¬†Media/Serum Cross Reference -¬EU GIBCO¬†Media/Serum Cross Reference - US

Media Expert Media Formulations Mediatech  - product cross reference chart MSDS/COA Search On-Site Stocking/Inventory Management

Poster Gallery Serum Planner

Videos cell-culture-video micropipette video

Invitrogen

Invitrogen Mammalian-Cell-Culture Primary Cell Culture

3D Cell Culture 3D-Cell-Culture Collagen I, Rat Tail AlgiMatrix Reduced Growth Factor Basement Membrane Matrix Stem Cell Research Stem-Cell-Research

Links

UMBC general TC method description

Wikipedia Tissue culture

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 UNSW Copyright Notice UNSW Copyright Notice Tissue Culture Lab 2 UNSW Cell Biology http://cellbiology.med.unsw.edu.au/units/science/lab0802.htm

Cell Analysis Lifespan Processes Birth Mitosis Except germ cells meiosis Death Apoptosis Programmed cell death Necrosis 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 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 Establishing a Cell Line Retinoblastoma Protein (Rb) 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 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