Experiment 6: Part 2: Development of Dictyostelium wild-type and myosin II mutant cells Over Agarose

Over Agarose Development of Dictyostelium discoideum

Agarose Preparation 

1)Make sufficient 1.5% agarose mixture in MCPB for the number of plates you will need.  The plates are good for several days after pouring if kept covered.  Heat in microwave, stopping to swirl the mixture occasionally, until agarose is completely dissolved.  Add 2 ml of this to a p60 dish and gently shake, rattle and roll the dish until the agarose is uniform, then let it harden on a level surface. 

Cells

1)You will need about 107 cells for each plate and about 5% fluorescent cells to mix with them. As in Part I, you can mix and spin or spin and mix.  

2)Triturate cells and centrifuge at 1200rpm for 5 minutes.

3)Re-suspend the pellet in 5mls cold MCPB.

4)Centrifuge again and resuspend the pellet in the desired volume.

5)Add 10 cells (plus GFP labeled cells) in 5ml MCPB to the agarose plates poured earlier.  Allow the cells to settle onto the agarose for 20-30 minutes.  They do not ever adhere to agarose as tightly as you have seen with plastic, so you have to be very gentle and patient.  Leave the cells on your benchtop microscope while they are settling so you can check if they are settled and attached without moving the plate too much. 

6)Gently remove the media.  First, remove all you can with your P1000 without tilting the dish.  Then remove the rest by tilting the dish slightly and sucking up the media with a pipette.  Blot the residual with a rolled up Kimwipe.  Do this gently so the cells and the agarose are not disturbed. 

Imaging

1)You can begin imaging immediately, or wait 4-6 hours and then begin. 

2)Gently place the plate on the microscope and begin the time-lapse, taking blue and white images once about every 60 seconds.  This is a good one to do at 4x because the wide field of view will help you see the population behavior.  You can up to 10 or 20x at some point if you want to focus in on slug migration, or aggregation.  On some of the microscopes, there is a PhL position in the condenser turret.  This will give you phase contrast with the 4x objective.  If you do not have one of the microscopes with 4x phase you can try closing the condenser aperture (the small tab just below the rotating phase turret

3)Development usually takes about 24 hours to complete.