Tissue Culture & Immunocytochemistry Part 1/2 Flashcards
Standard tissue culture condition?
- Humid
- 37°C,
- elevated CO2
- pH in physiological range (could use phenol red)
List some methods of aseptic technique in the lab
- tissue culture hood
- laminar flow, -ve pressure, filters
- alcohol (70% IMS)
- UV light sterilisation
- medium containing antibiotics (penicillin and streptomycin)
Define primary cell model
Derived directly from tissue
Define secondary cell model
Derived from sub-culturing primary culture
Define immoralised cell lines
Population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, can keep undergoing division
Differences between primary cells VS cell lines
- cellular purity
- no. of cells
- robustness of cells
- cell phenotype differentiated vs proliferative
- animal use
Methods of generating immortalised cell lines
- Isolate from naturally occurring cancer
- Introducing mutation that partially deregulates cell cycle
- Hybridoma production i.e. fusion of two cells
Examples of neuronal cell lines
- PC-12 neuroblastoma in rat adrenal medulla
- SH-SY5y neuroblastoma cells (dopaminergic characteristics)
- F11-somatic cell hybrid of rat embryonic dorsal root ganglion and rat neuroblastoma cell
NB: Non-physiological outgrowth and not post axotomy
Examples of non-neuronal cell lines
3T3 Mouse fibroblast
COS- monkey kidney
CHO- chinese hamster ovary
MDCK- dog epithelial
HeLa- Human epithelial (Henrietta Lacks)
L6- Rat myoblast
293t- human kidney (transformed)
Describe the process of sub-culturing cells
Dividing cells become confluent when they cover the bottom of the plate. Contact inhibition prevents them from continuing to divide -> become quiescient
If left, they become senescent
Describe the process of harvesting adherent cells
Cells washed with PBS (phosphate buffered saline)
PBS containing trypsin and EDTA is most common solution used
Proteolyitic trypsin cleaves the proteins holding cells together, aided w/ gentle agitation
EDTA chelates calcium and magneusium ions to inhibit cadherin and integrin-dependent cell-cell adhesion
Cells should be detached after 5-10 mins
Trypsin is neutralised now (otherwise it will digest the cells away)
Trypsin inhibitor OR (more routinely): plate is flooded w/ protein solution e.g. equal volume of 10% FCS solution
Centrifugation separates cells from trypsin solution
Unwanted solution is removed form cell pellet and discarded
Pellet can be resuspended in a suitable growth medium
Cell counting on haemocytometer gives cell density
Cells can be plated on fresh plates at the desired density
Examples of neuronal primary cell cultures
E16-18 or P0 cortical neurons
Adult DRG
P7 cerebellar granule neurons
E16-18/P0 hippocampal
P0 retinal ganglion cell
Describe how to culture primary cells
Culture on adherent substratum coated w/ poly-L-lysine, collagen or laminin
Either clutured in mixed culture w/ other cells, or purified
How can we increase the purity of the cell culture?
Fluorescence-acticated cell sorting (FACS): use of antibodies/dyes/genetic tagging to sort cell population
Immunopanning: use of cell-specific antibodies to sort the cell populaiton
Magnetic cell sorting: using antibodies and magnetic tagging to sort cell population
Neurite Outgrowth Assays
Permissive substrates: ______
Inhibitory substrates: ______
Neurite Outgrowth Assays
Permissive substrates: PLL/PDL or laminin
Inhibitory substrates: myelin, CSPGs or MAG
