Soft Agar Colony Formation Assay: A Method to Test Effects of Novel Compounds on Cancer Cell Proliferation

To begin, take an appropriate concentration of molten agarose in a conical tube and add a desired amount of warm culture medium to it. Gently invert to mix the contents. Add this mixture into each well of a six-well plate, and allow it to solidify. This is the bottom layer. Next, treat cancer cells with the compound of interest and add the required concentration of agarose.

Now, pour this mixture containing the desired number of cells per milliliter into each well. This is a cell-containing layer. Once the medium solidifies, incubate the plate at 37 degrees Celsius for a week. Prepare a feeder layer by mixing agarose to the medium and supplement this mixture with the compound of interest. Gently add this feeder layer into each well, allow it to solidify, and incubate the plate at 37 degrees Celsius.

Repeat this feeding procedure weekly to replenish the cells with the medium until colonies form. If the compound of interest is an inhibitor of tumorigenicity, it will suppress cells growth resulting in few colonies in the wells. In the following protocol, we will perform a soft agar colony formation assay to study the effect of PADI inhibitors on the tumorigenicity of breast cancer cells.

Begin this procedure by preparing 3% 2-hydroxyethyl agarose. Into a clean, dry 100 milliliter glass bottle, add 0.9 grams of 2-hydroxyethyl agarose followed by 30 milliliters of distilled water. Microwave the mixture for 15 seconds and gently swirl. Repeat this step until the agarose powder fully dissolves. Next, autoclave the solution containing bottle for 15 minutes.

Allow the agarose solution to cool down to room temperature before further use. Pre-warm several five milliliter and 10 milliliter pipettes in a 37 degrees Celsius incubator to prevent the agarose from solidifying in the pipette when handling. Partially loosen the bottle lid and microwave the pre-made 3% 2-hydroxyethyl agarose solution for 15 seconds. Then gently swirl the solution and microwave for another 15 seconds.

Be careful when swirling the agarose solution because the solution rises up when exposed to air and can spill over. If there is residual solid gel in the bottle, microwave for a few more seconds. Keep the bottle containing the agarose solution in a 45 degree Celsius water bath during the next steps to prevent the agarose solution from solidifying prematurely.

Next, transfer 12 milliliters of warmed media using the pre-warmed pipettes into a sterile 50 milliliter conical tube. Immediately add three milliliters of the 3% agarose solution and gently invert the conical tube to mix the agarose with the media. Then gently add two milliliters of this mixture into each well of a six-well culture plate without forming any air bubbles. Incubate the six-well culture plate horizontally on a flat surface at four degrees Celsius for one hour to allow the mixture to solidify.

After the mixture solidifies, place the plate into a 37 degrees Celsius incubator for 30 minutes. The bottom layer is now ready for use.

A difficult aspect of this procedure is to make sure that all cells are distributed equally and that the melted agarose gel do not solidify before addition to each well, to ensure that the cells are mixed in the media before adding the liquid agarose gel. Additionally the pipettes are pre-warmed beforehand to avoid the solutions from solidifying while handling.

To prepare the cell containing layer, first trypsinize MCF10DCIS cells and dilute them to a cell concentration of 40,000 cells per milliliter. Then take eight milliliters of media using pre-warmed pipettes, and transfer into a sterile 50 milliliter conical tube. Immediately add two milliliters of 3% agarose to the conical tube, and gently invert to mix the agarose with the media. Avoid forming any bubbles.

Next, take two milliliters of the cells and treat them with two micromolar BB-chlor-amidine in DMSO, or DMSO alone as the control. Following treatment, mix the cells with a 0.6% agarose in a one to one dilution to make a final concentration of one micromolar BB-chlor-amidine. Then take one milliliter of the cell-agarose mixture and gently add onto the bottom layer of the six-well culture plate.

Place the six-well culture plate horizontally on a flat surface at four degrees Celsius for at least 15 minutes to allow the top layer to solidify. After the mixture solidifies, place the plate into a 37 degrees Celsius incubator for a week before adding the feeding layer. Begin feeding layer preparation by microwaving the pre-made 3% 2-hydroxyethyl agarose solution as before, and equilibrating the agarose solution bottle in a 45 degrees Celsius water bath.

Mix one milliliter of 3% agarose solution with nine milliliters of warm media into a 50 milliliter conical tube, and gently invert to mix the agarose with the media. Avoid forming air bubbles. After treating the mixture with BB-chlor-amidine, gently add one milliliter of the mixture into each well of the six-well culture plate containing the bottom and soft layers. Then place the six-well culture plate horizontally on a flat surface at four degrees Celsius for at least 15 minutes to allow the mixture to solidify.

After the feeder layer solidifies, place the plate into a 37 degrees Celsius incubator. Repeat this feeding procedure weekly by overlaying one milliliter of 0.3% agarose medium treatment solution onto the existing feeder layer to replenish the cells with new media until colony formation is observed.