WangLab:EGF Experiment

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Time for experiment

  • 30-60 minutes

EGF Experiment

  1. 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.
  1. Start Metafluor
  1. Open a CFP-YFP-FRET protocal
  1. Use FRET-open to locate a good cell.
  1. Starting focusing to adjust focusing and exposure time.
  1. Configure-> Aquisition to set exposure time.
  1. 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.
  2. Aquire 5 images at 60 seconds intervals.
  1. 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.
  1. Start Aquiring at 30 seconds intervals for 20 cycles and the at 60 seconds intervals for 20 cycles.
  1. Adjust Focus if necessary.

Note

  • The same protocol can be used for pervanadate experiments by changing EGF to pervanadate.
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