Immunocytochemical Study of Apoptosis Signaling In Nb2 Lymphoma Cells

• Main Author: Guanzon, Angelo P.
• Format: Others
• Published: VCU Scholars Compass 1998
• Subjects: Physiology
• Online Access: http://scholarscompass.vcu.edu/etd/4691; http://scholarscompass.vcu.edu/cgi/viewcontent.cgi?article=5768&context=etd

In the absence of mitogen, administration of Dexamethasone (Dex) induces apoptosis, or programmed-cell death in the Nb2 lymphoma cell. Addition of prolactin (Prl), on the other hand, blocks this effect. As a model for apoptosis, we were able to investigate this Dex-Prl interaction by means of a morphological approach: one that could be visualized under a light microscope. This approach allowed us to achieve several aims. First, we were able to develop a method of cell quantification befitting a morphological study based upon the hemacytometer. Second, with Trypan Blue exclusion, we were able to verify Dex/Prl-responsiveness in the Nb2 cells. Third, we were able to refine and characterize the TUNEL (Tdt-dependent dUTP-biotin Nick End Labeling) assay as a means of detecting apoptosis in both log phase and synchronized cells. With the synchronized cells, we observed that the time frame of apoptosis onset as measured by Trypan Blue and the TUNEL assay, occurred between 6 and 8 hours. Fourth, using immunocytochemistry (ICC), we were able to characterize and establish specificity of affinity purified polyclonal rabbit antibodies directed against the signal proteins, glucocorticoid receptor (GR), STATSb, NFkB and lkBα. Fifth, we were able to examine how these signal proteins changed in response to Dex treatment using ICC. According to our results, the percentage of positively stained cells for each of these signal proteins remained constant for each time point and treatment.