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

1. Preparation of 3% 2-Hydroxyethyl Agarose

1. Into a clean, dry 100 ml glass bottle, add 0.9 g of 2-hydroxyethyl agarose (Agarose VII) followed by 30 ml of distilled water.
2. Microwave the mixture for 15 sec and gently swirl. Repeat this step at least three more times until the agarose powder fully dissolves.
3. Autoclave the solution-containing bottle for 15 min.
4. Allow the agarose solution to cool down to RT before further use. Store the solution at RT.

2. Preparation of the Bottom Layer: 0.6% Agarose Gel

1. Pre-warm several 5 ml and 10 ml pipettes in a 37 °C incubator to prevent the agarose from solidifying in the pipette when handling.
2. Partially loosen the bottle lid and microwave the pre-made 3% 2-hydroxyethyl agarose solution for 15 sec. Then, gently swirl the solution and microwave for another 15 sec.
   CAUTION: Be careful when swirling the agarose solution because the solution rises up when exposed to air and can spill over.
3. If there is residual solid gel in the bottle, microwave for a few more seconds.
4. Keep the bottle containing the agarose solution in a 45 °C water bath during the next steps to prevent the agarose solution from solidifying prematurely.
5. Warm MCF10DCIS media in a 37 °C water bath.
   NOTE: MCF10DCIS media consists of DMEM/F12, 5% horse serum, 5% penicillin streptomycin.
6. Transfer 3 ml of the 3% agarose solution using the pre-warmed pipettes into a sterile 50 ml conical tube.
7. Immediately add 12 ml of warm MCF10DCIS media and gently invert the conical tube to mix the agarose with the media. Try not to form any bubbles as it will interfere with the colony counting later.
8. Gently add 2 ml of this mixture into each well of a 6-well culture plate without forming any air bubbles.
9. Incubate the 6-well culture plate horizontally on a flat surface at 4 °C for 1 hr to allow the mixture to solidify.
10. After the mixture solidifies, place the plate into a 37 °C incubator for 30 min. The bottom layer is now ready for use.

3. Preparation of the Cell-containing Layer: 0.3% Agarose Gel

1. Trypsinize MCF10DCIS cells and dilute them to a cell concentration of 4 x 10⁴ /ml.
2. Take 2 ml of the 3% agarose using pre-warmed pipettes and transfer into a sterile 50 ml conical tube.
3. Immediately add 8 ml of MCF10DCIS media to the conical tube and gently invert to mix the agarose with the media. Avoid forming any bubbles.
4. Take 2 ml of the MCF10DCIS cells (4 x 10⁴ /ml) and treat with BB-CLA (0 µM (DMSO) or 1 µM).
5. In a 1:1 dilution, mix the cells with the 0.6% agarose.
6. Take 1 ml of the cell-agarose mixture and gently add onto the bottom layer of the 6-well culture plate (2 x 10⁴ cells/ml).
7. Place the 6-well culture plate horizontally on a flat surface at 4 °C for at least 15 min to allow the top layer to solidify.
8. After the mixture solidifies, place the plate into a 37 °C incubator for a week before adding the feeding layer.

4. Preparation of the Feeder Layer: 0.3% Agarose Gel

1. Microwave the pre-made 3% 2-Hydroxyethyl agarose solution for 15 sec. gently swirl the solution and microwave for another 15 sec.
2. Equilibrate the agarose solution bottle in a 45 °C water bath.
3. Warm the MCF10DCIS media in a 37 °C water bath.
4. Mix 1 ml of 3% agarose solution with 9 ml of warm MCF10DCIS media into a 50 ml conical tube and gently invert to mix the agarose with the media. Avoid forming air bubbles.
5. Treat the mixture with BB-CLA (0 µM (DMSO) or 1 µM).
6. Gently add 1 ml of this mixture (without forming bubbles) into each well of the 6-well culture plate containing the bottom and soft layers.
7. Place the 6-well culture plate horizontally on a flat surface at 4 °C for at least 15 min to allow the mixture to solidify.
8. After the feeder layer solidifies, place the plate into a 37 °C incubator.
9. Repeat this feeding procedure weekly by overlaying 1 ml of 0.3% agarose/medium/treatment solution onto the existing feeder layer to replenish the cells with new media until colony formation is observed.
   NOTE: Agar in the soft and feeder layers is very soft and, therefore, the added nutrients from the feeder layer will readily diffuse into the cell-containing layer to reach the cells.