Question

Design a microscopy experiment (widefield) testing the effect of Vertiporfin on cell death (step by step). What do we look for? which colour are we running, how long are we running it for?

Note: don't worry about the microscope, just design an experiment for staining of cells so it can be looked under widefield microscope

Experimental Themes, Treatments

Growth Surface Interactions - Cell adhesion and spreading : Zetag (cationic polymer), Fibrinogen,

Cell shape, size and cytoskeleton : Verteporfin, manitol

Cell death : Vertiporfin, Manitol, Curcumin, Berberine, Epirubicin

Fluorescent Probes (Reagents to choose from) WGA488, WGA568, phalloidin CF488, phalloidin CF568, Mitotracker Orange, Annexin V AF488, Annexin V 568

Experimental layout (Maximum 2 X 6 well plates)

Autofluoresence control	single colour control	single colour control	single colour control	no treatment	treatment -Treatment type Time
treatment -Treatment type Time	treatment -Treatment type Time	treatment -Treatment type Time	treatment -Treatment type Time	treatment -Treatment type Time	treatment -Treatment type Time

Answer 1

Maintain a particular cell line and observe them for any contamination or degradation. Take an appropriate number of cells to seed them in the 6 well plates with media. Keep the cells in incubator and observe for a night. Next day give these seeded cells treatment with the aforementioned drug-  Vertiporfin. The cells are then observed upto 72 hours at the intervals of 24 hours. In MTT assay, the cells are after 24 hours are added with the dye MTT. Cells that are alive, have high metabolism and would reduce the dye to produce purplr color. If the cells are dead they wont be able to produce the purple color due to low reductive power.