Anchorage-Independent Growth Assay

(Soft Agar Colony Formation)

For immortalized fibroblasts done in 60 mm plates:

The bottom layer of soft agar is 0.4% agarose in DMEM w/5% FBS-HI

Make a 2X stock of DMEM from powder, add pen/strep and 10% FBS-HI

If you have a selection drug, include it in the 2X medium stock.

May also use lower levels of serum to look at serum-dependence.

Use agarose as sometimes agar prohibits growth and/or gives a very high background.

Make a 2X 0.8% agarose stock in sterile water and autoclave. Store at room temperature.

To pour bottom layer:

For 60 mm plates, bottom agarose layer will be 2.5 ml total

Set a water bath to 50^oC

Microwave agarose stock to liquefy and then put bottle in 50^oC water bath

Set up sterile 50 ml conical tubes with 2X medium and put in 37^oC water bath.

When plates are ready (labeled, etc), bring 50 ml conical tubes into hood, then agarose stock, adding agarose stock 1:1 volume to 2X medium and quickly pouring into 60 mm plates. Swirl plates so agarose hardens evenly. Allow to cool and harden completely (but not dry out). Can store these plates at 4^oC (upside down and in the dark/wrapped in foil) for up to two weeks prior to use. Plates must be made at least the day before use.

The top layer is 0.3% - 0.4% agarose plus cells in DMEM w/5% FBS-HI

Use the same 2X medium stock as above.

If using an inducible expression vector, include inducing agent in top layer medium (and all additional medium added to keep plates from drying out).

Use the same 2X agarose stock as above. Certain cell types are sensitive to the agarose percentage and need to determine if 0.3% or 0.4% agarose is best suited to cell type in use.

To pour top layer:

For 60 mm plates, top layer will be 3 ml total.

Set a water bath to 45^oC

Microwave agarose stock to liquefy and then put in 45^oC water bath

As cells will be included in this layer, don’t want to have agarose at too high a temperature that may damage cells

Set up sterile conical tubes with 2X medium and put at 37^oC

Pre-warm plates by placing in 37^oC incubator

Prepare cells, want densities ranging from 10³ – 10⁴/per 60 mm dish and done in duplicate.

Bring 2X medium and hot agarose into hood, add cells to medium and mix quickly, then add agarose, mix quickly and pour over bottom layer of agarose, swirling to distribute evenly. Allow to cool and harden (but not dry out). Add 1 ml 1X growth medium to the tops of plates so as not to let agarose dry out.

Look at plates every day to make sure they have not dried out and add 1 ml 1X growth medium to the plates when necessary.

Watch for growth of colonies and score at 3 – 4 weeks of growth.