You have been asked to replicate the following experiment and validate results b

Question

You have been asked to replicate the following experiment and validate results before attempting a similar experiment. You need to: Prepare all needed reagents Prepare Stock Solutions Prepare Working Stocks And describe the purpose of the control vehicle C16-ceramide on germ cell apoptosis: Fresh stock solution of 5 mM C16-ceramide (Cayman Chemical, 24696-26-2) was prepared by dissolving desiccated C16-ceramide in ethanol containing 2% dodecane (vehicle) at 37°C. To induce germ cell apoptosis, the stock solution was diluted in M9 buffer (22 mM KH2PO4, 42 mM NaHPOs, 85.6 mM NaCI, 1 mM MgSOs) to the desired concentration and microinjected into the posterior gonad of young adult hermaphrodites. Controls received vehicle (1x10 3-5x102 dilution in M9 buffer). Germ cell corpses in the posterior gonad were counted at the indicated times after injection. For restoration of radiation-induced germ cell apoptosis, worms were irradiated immediately following ceramide injection. For these studies, sub- lethal doses of Cio-ceramide were titrated against restoration of the radiation response; while 0.001 HM gonadal ceramide (calculated as below) was ineffective, complete restoration was achieved at 0.005 M gonadal ceramide.

Explanation / Answer

Ceramide engagement in adoptotic pathways is very controversial

Ionizing radiolation-induces adoptosis of germ cell was obliterated on inactivation of ceramide

Radiation induced increase the concentration of ceramide in mitochondria

Inactivation of CEP-1 pathways and accumulation of EGL-1 and ceramids synthatase-mediated generation of ceramide

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