Soft agar assay

Soft Agar Assay Protocol

Authors:

Shi Yun Yeo, Yoko Itahana, Koji Itahana

Materials

Procedure

1.       Make a suspension of 3% agarose in PBS in a glass bottle by mixing (for example, 3g of agarose powder in 100 mL of PBS).

2.       Put a glass bottle in an autoclave and heat the suspension to 121^oC for 15mins. This process solubilizes and sterilizes the agarose suspension. After sterilization, keep agarose gel in a liquid state by placing the bottle containing the gel in a 60^oC water bath.

3.       Make DMEM media containing 15% FBS. Ensure that the temperature of the media is kept at 37^oC. Cold media will promote premature aggregation of gel.

4.       Make a bottom agar containing 1% agarose. For a 6-well plate, mix 5 mL of autoclaved 3% agarose in PBS with 10 mL of media containing 15% serum.

5.       Pipette 2 mL of a bottom agar per well, ensuring that bubbles are not introduced in the process.

6.       Allow the bottom agar to solidify at room temperature.

7.       In the meanwhile, make a top agar containing 0.8% agarose. For a 6-well plate, mix 6 mL of autoclaved 3% agarose in PBS with 16.5 mL of media containing 15% serum in a 50-mL plastic tube. Always prepare gel where there is at least 20% excess to facilitate pipetting.

8.       Keep the gel at 37^oC by placing the tube in a water bath to prevent premature solidification of gel.

9.       Trypsinize cells, add medium containing FBS to neutralize trypsin, keep cells on ice, and count cells.

10.   Put the necessary numbers of cells suspended in media containing 15% serum in a 15-mL tube and mix with the same volume of a top agar carefully by minimizing the introduction of bubbles. At this step, % of agarose becomes 0.4%. HMEC^{TERT/ST/ER-RasV12} cells need 20,000 cells in 2 mL per well. BJ cells need 40,000 cells in 2 mL per well. NIH-3T3 cells need 20,000 cells in 2 mL per well.

11.   Plate 2 mL of a top agar containing cells per well in a 6-well plate.

Tip: Suck up 7 mL, keep the last 1 mL with bubbles in pipet.

12.   Put it IMMEDIATELY for 20 min at 4^oC to let solidify. Note: Do NOT stack the plates as the middle plates will not solidify.

13.   For HMEC^{TERT/ST/ER-RasV12} cells, agar was topped up with 1 mL DMEM containing 500 nM of 4-OHT for the first 3 days, and then replaced with DMEM containing EGF (5 ng/mL), insulin (5 μg/mL), hydrocortisone (500 ng/mL), and 4-OHT (500 nM). The top media was replaced after one week. For BJ cells, agar was topped up with 1 mL DMEM containing 500 nM of 4-OHT and 10% FBS for the first 1 week. After 1 week, the top medium was replaced with DMEM containing EGF (5 ng/mL), insulin (5 μg/mL), hydrocortisone (500 ng/mL), 4-OHT (500 nM), and 10% FBS. For NIH-3T3 cells, agar was topped up with 1 mL DMEM containing 500 nM of 4-OHT and 10% FBS. The top media was refreshed after one week.

14.   After 2 weeks, take out a top media, add MTT staining solution (5 mg/mL MTT in DMEM media containing 10% FBS,  500μl for each well), and stain at 37^oC for 1hr.

15.   Photograph cells with a stereomicroscope (SZX16, Olympus) and count colonies. Counting of colonies was automated using a MATLAB script based on the intensity and size of stained colonies.