I have seen most publications using 0.005-0.01% crystal violet for staining of soft agar colony assay. Is methylene blue interchangable? If yes, how should I go about performing the stain? More details would be appreciated. Thanks!
Hello, I have stained soft agar colonies with methylene blue before. Here is my protocol: Soft agar assay was done in 6 cm dishes. A bottom layer of 4 ml, 0.8% agarose (Lonza, Catalog No. 50 000) in complete medium, was made. On top, a layer containing 10 000 cells mixed in 3 ml 0.35% agar (Difco, Catalog No. 214220, Becton, Dickinson and Company, Spark, USA) in complete medium was spread. After 2 days incubation, another layer of top agar in complete medium was added. Colonies were visualized by staining with 0.005% Methylene blue and photographs were taken in a dissection microscope.
Hope this is helpful and good luck with your experiments.