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CHARACTERIZATION OF CELLS
In situ hybridization
Autoradiography
General procedures
Visualization of results
Revision
A primary culture refers to a culture from the time of isolation
until its first subculture.
CULTURE HETEROGENETY
Most individuals began as a single cell and are therefore the result of clonal
expansion in vivo.
Cell Cloning is the generation of a colony from a single cell, and subculture
of such a colony would give rise to a cell strain.
Cloning is used
The dilution
105.6 /101 = 104.6 (dilute 40,000 times to get 10 cells per ml)
105.6/101.7 = 103.9 (dilute 8,000 times to get 50 cells per ml)
105.6/102 = 103.6 (dilute 4,000 times to get 100 cells per ml)
105.6/102.3 = 103.3 (dilute 2,000 times to get 200 cells/ml)
ISOLATION OF CLONES
• Trypsin, purified is best and carried out at 4oC, can carry out
trypsinization at 37oC.
• Conditioned medium- medium that has been used for the growth
of other cells acquires metabolites, growth factors and matrix
products
iii. Transformed
i.e. finite or continuous cell line
have malignancy properties
v. Morphology :
appearance and patterns of the cells
Disadvantages;different culture conditions can change the morphology
Use : inverted microscopy, staining methods,
NORMAL HYBRIDIZATION
• separating it on a gel,
G C T A
Complementary
nucleotide bases
probe
CTTAGGTCAGTAA
HYBRID Target gene
GAATCCAGTCATT
Hybridizatiion ; chemical reaction between the probe and
the DNA or RNA to be detected
Examples of some use of hybridization
Radiolabelled-RNA
OR
Radiolabelled-RNA probe
5’ transcripts 3’
RNA
UCCAAUGGCUUAUUUCCCUA
3’
DNA 5’
3. Prehyvridization
4. Hybridization
-hybridization buffer
–formamide-helix destabiliser
Reduces the melting point of the hybrid – lower temperature helps to
preserve tissue architecture
Dextran sulphate -increaes the concentration of the probe by volume exclusion,
Therefore reduces hybridization time
Fig 2. The presence of virus in the nucleus of the infected cell stained with
rabbit anti DNA-RNA conjugated with FITC. The DNA infected virus
in the cell nucleus is stained fluorescent green