Experiment 3  Part 2: Is there a difference between wild-type and myoII- mutant movement on a surface



1. Get your microscope set up so the focal plane is correct for cells on the bottom of a petri dish.

2.Add 3.5ml of HL5 to a 60mm dish and place it on the stage of the microscope.  Get your time lapse parameters set up in advance.

3.Now add about 1 X105 cells to the dish and gently swirl to disperse.  Alternatively, you could prepare the cells in media and add it to a clean dish and quickly put it on the stage.

4.Image the cells over time.

5.The cells will gradually settle to the bottom, attach and begin to move.


Questions

1.How long does it take for the cells to finish settling?

2.How long does it take for them to begin moving?

3.Once they start moving, do they stay at the same speed or increase in speed of vary in speed?

4.Is there a difference between the mutant and wild-type in the behavior (does the process look the same?) and quantifiable parameters (speed, persistence, etc.)



How long after plating?

Cells settle to the bottom

How long do cells reattach to the plate?

How long to begin moving?

Are they grown in fresh media? How does this change the osmolarity and media components (growth factors) affect behavior?

Does it matter that they are on a new plate? Do cells condition the surface as they grow?Where is each cell at in the cell cycle?



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