Cell Studies
Why doing cellular studies
• The physicochemical environment can be
accurately controlled(PH,O2,CO2)
• Sample is homogenized and well
characterized
• It can be cost saving
How would this relate to the project
• This would provide us with the data
– Make more accurate model of the system
– Better chance of controlling the HSP
expression
– Maybe species and tissue dependent
How to get started
• For this we need normal canine cells from
prostate and neighboring tissues
– Bladder
– Rectum
– Urethra
Cell lines
• Some commercially available
– Not in our case
• Getting them from other research institutes
– Not successful
• Deriving them from the tissue
Cell Isolation
• Mince or cut the isolated piece of tissue into 2-4
millimeter pieces with sterile scissors or scalpel.
• Add appropriate amount of enzyme(s) and
incubate at optimum temperature (usually 37°C)
for appropriate time, mixing intermittently.
• Gently disperse the cells by pipeting.
• Quantitate cell yield and viability.
• Seed cells for culture, if required.
• Separate cells of interest.
Cell Isolation
Cell Culture
• Cell cultures are derived from either
primary tissue explants or cell
suspensions.
• Primary cell cultures typically will have a
finite life span in culture whereas
continuous cell lines are, by definition,
abnormal and are often transformed cell
lines.
Culture Media
Western Blot
• 1) Proteins are separated by gel
electrophoresis, usually SDS-PAGE.
• 2) The proteins are transferred to a sheet of
special blotting paper called nitrocellulose.
The proteins retain the same pattern of
separation they had on the gel.
• 3) An antibody is then added to the solution
which is able to bind to its specific protein.
The antibody has dye attached to.
TVT
• TVT is a naturally occurring transplantable tumor that is
typically propagated and maintained among the dog
population by coitus and the social behavioral trait of
smelling and licking of the external genitalia. Although the
tumors are located most commonly on the external
genitalia (prepuce, penis, vulva, and vagina), they also
have been reported extragenitally in the skin, lips, buccal
and nasal mucosa, and less frequently in the tonsils, liver,
pancreas, spleen, lung, kidney, and inguinal and
mesenteric lymph nodes. Tumors begin as nodules in the
submucosa or subcutis and typically undergo spontaneous
regression in 4-6 months. Tumor regression is commonly
associated with infiltrating lymphocytes composed primarily
of T cells.