Melt 1.2% Agar (molecular biology grade, low melt temp), cool in 42°C water bath. Warm 2x medium + 20% FBS + 2x antibiotics in 42°C water bath. Allow at least 30m for temperature to equilibrate.
Mix equal volumes of two solutions to give 0.6% agar + 1x medium + 10% FBS + 1x antibiotics.
Add 1.5 mL/35 mm Petri (not tissue culture) dish, allow to set in culture hood. The plates can be stored at 4°C for up to 1 week.
Melt 0.6% Agar (molecular biology grade, low melt temp), cool in 42°C waterbath (it is important not to exceed 42°C, otherwise cells will be killed). Also warm 2x medium + 20% FBS + 2x antibiotics to the same temperature.
200-5000 cells/35mm plate are required, therefore adjust number of cells to add into 750 µL of 2x medium (remember this medium with cells will be diluted 1:2 when mixed with 0.6% agar). For example, for 1 – 35 mm dish at 5000 cells/plate, the concentration of cells in 2x medium will be 6666 cells/mL. Keep at 37°C prior to plating.
For each 35 mm dish, mix 750 µL of cells in 2x medium + 750 µL of 0.6% agar to give you 1.5 mL of 0.3% agar in 1x medium containing 5000 cells, then immediately plate on top of base agar. Assays are usually done in triplicate, so account for 3 wells/condition in calculations; best to make 4 wells’ worth for safety.
Incubate assay at 37°C in humidified incubator from 10 up to 30 days, depending on cell growth. To decrease risk of contamination, place plates inside a 15 cm tissue culture plate, with an open 35 mm plate of sterile water in the middle for hydration.
Optional: Stain plates with 0.1% Crystal violet in 10% ethanol for 30 minutes, de-stain using lots of dH2O rinses, then count with dissecting scope.