Time for experiment
- 30-60 minutes
- Set the cell culture dish on the stage of microscope.
- Change the dish cover to a piece of flat cover glass.
- Make sure the dish is securely settled on the rack of the stage.
- Make sure the 40x objective touches the bottom of dish.
- Start Metafluor
- Open a CFP-YFP-FRET protocal
- Use FRET-open to locate a good cell.
- Starting focusing to adjust focusing and exposure time.
- Configure-> Aquisition to set exposure time.
- Aquire one image, define regions.
- Enable background subtraction of a constant
- The constants are chosen by image histograms.
- Define regions for tracing change of FRET.
- Aquire 5 images at 60 seconds intervals.
- Pause aquiring.
- Adding 20 ul x 20 ug/ml EGF to 2 ml dish (200ng/ml), to the edge of dish.
- Pipet (big, 1000ul) up and down 3 times to mix well.
- Be careful not to touch anything or cause the cell culture dish to move.
- Add events
- Events -> EGF -> mark events.
- Start Aquiring at 30 seconds intervals for 20 cycles and the at 60 seconds intervals for 20 cycles.
- Adjust Focus if necessary.
- The same protocol can be used for pervanadate experiments by changing EGF to pervanadate.