What I am doing is using Methyl Cellulose to do the anchorage independt assay with the bottom of the 96-well plate coated with polyheme to prevent cell adhesion. However, after I have repeated the assay many times, I found what caused the problem was the actual coating of the bottom of the plate went wrong.
Ideally, the coating shouldn't give any background at any stage after coating. However, when polyheme is tried (I always led it try for around 1hr. Then I found it actually dried up in 30-35min. Before it's dried, the bottom of the well looks smooth and right after the 30-35min drying the well suddenly becomes completely dried and when I looked down the microscope, I observed an opaque patterning instead of a smooth and relatively transparent background. I think it's due to the lack of adhesion prevention, some of the suspending cells in the methyl celluose sunk to the bottom and divided like normal cell culture.
Does anyone know how to solve this polyheme problem? Thanks a lot. Here is my protocol for this assay:
Preparation of Polyhema:
Dissolve 0.084g polyhema powder in 700µl 95% EtOH. It gives 700µl 10x polyhema solution. Leave in 60°C waterbath, votexing occasionally for hours and leave it in waterbath O/N. The next morning add 6.3ml 95% EtOH to dilute it down to 1x (final conc. 12mg/ml). Put it back to 60°C in waterbath, votexing occasionally for a couple of hours before plating.
Add 50µl to each well except peripheral normal medium only wells. Air-dry in the hood with lid off for 1hr.