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

Answer 1

Experiment

Prodedure

1. Grow cells onto a glass cover slip in 24 well plate at a concentration of 25000 cells per well.

2.next day after cell atachment, treat cells of one well with vertiporfin and use one well as control where no treatment will be given.

3. After the required incubation, centrifuge cells in the plate centrifuge for 15 mins at 3000 rpm.

2. After the dead cells settle down, remove the media and wash cells with phosphate buffer saline(pbs) two times.

3. Add propidium iodide (PI) 10ug/ul made in PBS to the cells and incubate in dark for 20 min.

4. After the incubation wash cells with PBS twice.

5. Pick up the glass cover slip (both treated and control) with a forcep and put it in an inverted fashion on a glass slide separately.

6. place the glass side inverted under 40X objective of the microcrope and excite with 591 laser to view PI staining(red). To view cells without stain use brightfield.

Result: Dead cells will take up the PI stain and will be seen red. PI is impermeable to live cells but dead cells whose cell membrane is compromised takes up the PI stain.