Basic chemotaxis experiments

Grow 3x D. discoideum / K aerogenes clearing plates for 36h.

Harvest & collect D. discoideum at 1.5 to 3x10⁷ cells/ml in development buffer (DB, dictybase). 

Centrifuge at 300g for 3 min, remove supernatant and resuspend pellet in DB 3 times.

Shake DB/cell mix for 1.5hr in a conical flask. 

Pulse cells with 300nM cAMP for 4.5 hours at 6-min intervals, bringing them into an aggregation competent state.

Take pulsed cells, pellet and resuspend at 2.5x10⁶/ml in outer well medium*.

Load Insall chamber with centre well medium**.  Close with 22x22x0.2mm coverslip. 

Drain outer well and refill with cells in outer well medium. 

Image on microscope (in our case, this was an inverted Nikon light microscope, using phase contrast with a 10x objective at 4 frames/min). 

*   For an imposed gradient, the centre well medium is 1 uM SpcAMPS/DB. For a self-generated gradient, it is 10 uM cAMP/DB.

** For an imposed gradient, the outer well medium is DB. For a self-generated gradient, it is 10 uM cAMP/DB.

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