Answer:
Explanation:
Null Hypothesis: Their will be no effect of the caffeine on the mitosis.
Hypothesis: Caffeine will inhibit the SHSY5Y cell division.
Dependent variable: Various concentration of the caffein compound.
Independent Variable: temperatures of the experimental condition. If their will be any effect of the temperature in mitosis then it will also affect the mitosis of control cells.
Experimental design.
Culture SHSY5Y neuronal cell line in the T-75 culture flask in CO2 incubator at 37 degree celsius. Next day, add cells from T-75 flask in the 24 well plate and allow cells to adhere to the well by incubating plate in CO2 incubator at 37 degree celsius. Add the compound caffeine at different concentration (say 1 microgram, 2 microgram, 3 microgram, 4 microgram) and incubate the plate for 24 hour. Determine the mitotic cells by following assay.
Assay to determine mitotic cell.
The reagent succinimidyl ester of carboxyfluorescein diacetate (CFSE) binds to cytoplasmic proteins. it is fluroscent reagent. And when cell divide due to mitosis, cytoplasm will also divide and hence, the protein which stained with the CFSE reagent will also goes in the daughter cell. Then, the fluorescence of the stained cells can be read on the plate reader.
Prepare the graph of the fluorescence values (Y-axis) verses different concentrations of the caffein (X-axis) and determine the slop of curve to know the rate of mitosis.<u>
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<u>Following test and control samples will be used for the assay.</u>
1. SHSY5Y cell line with added caffeine. It is test sample.
2. SHSY5Y cell line added with buffer. This important to know behaviour of cells under experimental conditions.
3. SHSY5Y cell line with solvent used to dissolve compound caffeine. It is important to know the toxicity or any effect of solvent on the mitosis.
