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.