I'm using LMP Agarose (AgarPlaque Plus Agarose, BD Bioscience) for the assay.
Lower layer: 0.5%
Upper layer: 0.4%
I'm trying to overcome few problems with the soft Agar assay:
1) Althoug I place the plate at 4C for 20min, most of the cells concentrate on top of the lower layer.
How could I overcome the cell spreading problem?
Maybe by adding reagent that would give higher Viscosity to the upper layer and by that maintain the cell scattered evenly in the upper matrix until Gelation??
2) After colonies appearance I want to use MTT Colorimetric method to quantify the live cells. How could I dissolve the solid matrix w.o. harming the mitochondrial Enzymatical activity?
I hope that simple solutions are avilable to solve those problems?
Thanks in advance,