Control samples for use as standards for evaluating apoptosis in a selected tissue
a technology of control samples and tissue, applied in the field of medicine and pharmacological research, can solve problems such as limitations of each of these methodologies, tissue atrophy or hypertrophy, autoimmune disease or degenerative disorder,
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example 1
Thymic Apoptosis in Microgravity Culture
[0055] Materials and Methods:
[0056] The following media and solutions were prepared: [0057] 1. Phosphate buffered saline (PBS) for rinsing tissue. [0058] 2. Control media, consisting of RPMi 1640 containing 15% heat-inactivated fetal bovine serum, 100 μg / mL penicillin, 100 μg / mL streptomycin, 100 μg / mL L-glutamine, and 2.5 μg / mL amphotericin B. [0059] 3. Experimental media, consisting of control media containing 1 μM dexamethasone.
[0060] Two microgravity bioreactors (Synthecon, Inc., Houston Tex.) were utilized; one containing control media and one containing experimental media.
[0061] The thymus was removed from two Lewis rats, washed in cold PBS to remove blood, and stripped of excess tissue. Thymus tissue was cut into sections of about 3 mm3, while in cold media.
[0062] The bioreactors were filled with control (CON) or experimental (DEX) media. Approximately 24 thymus pieces were placed in each bioreactor, for incubation at 37° C. at a r...
example 2
Apoptosis of Several Tissue Types in Microgravity Culture
[0068] The experiment described in Example 1 was repeated with each of the following tissue types: heart, kidney, liver, spleen, lymph node and skin. Results paralleled those observed and set forth above for thymic tissue.
example 3
Detection of Apoptosis Markers in Microgravity Cultured Tissues
[0069] The experiments described in Examples 1 and 2 were repeated with one or more of the following tissue types: thymus, heart, kidney, liver, spleen, lymph node and skin. Markers of apoptosis were detected immunohistochemically in the treated tissue, and / or by Western blot in culture fluid. Specific apoptosis marker proteins assayed for included annexin, one or more caspases, and Fas / FasL.
[0070] Results showed that markers of apoptosis increased in a graded fashion, similar to the results observed from TUNEL assays of the tissues. In addition, the marker proteins accumulated in the culture fluid in a similar graded fashion over time.
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