Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

The overall goal of this procedure is to quantitatively determine the effect of treatment on the ability of cells to proliferate in semi-solid matrices as enhanced ability is a hallmark of cell tumor genicity using the soft agar assay tumor genicity is measured through a cell's ability to form colonies within a semi-solid AROS gel. Since non transformed cells are unable to rapidly propagate in this environment, the semi-solid AROS gel is constructed by first making a 0.6%AROS bottom layer. Next, a cell containing 0.3%AROS gel layer is added above the bottom layer.

In this assay, the experimental cells are treated with an inhibitor of peptidyl, arginine, dase, or pad enzyme developed by Dr.Subramanian. Every week, an additional feeding layer is added, which is a 0.3%aros gel with or without treatment. The final step is to count the number of colonies larger than 70 micrometers for analysis.

Ultimately, inverted microscopy is used to show the difference in the colony number between the control and the treatment group. Hi, my name is Achi Huta. I am a graduate student in Dr.Scott Kros lab at the Baker Institute for Animal Health.

At Cornell University, soft tiger colony formation assay can help answer key questions in cancer biology field, such as assessing the tumor genicity of a wide range of cancer cells with regards to their sensitivity to drugs, hormones, and a multitude of other treatment conditions. Begin this procedure by preparing 3%two hydroxyethyl aros into a clean, dry 100 milliliter glass bottle. Add 0.9 grams of two hydroxyethyl aros, followed by 30 milliliters of distilled water.

Microwave the mixture for 15 seconds and gently swirl. Repeat this step until the AROS powder fully dissolves. Next, autoclave the solution containing bottle for 15 minutes.

Allow the agros solution to cool down to room temperature. Before further, use Prewarm several five milliliter and 10 milliliter pipettes in a 37 degrees Celsius incubator to prevent the aros from solidifying in the pipette. When handling partially, loosen the bottle lid and microwave the pre-made 3%two HYDROXYETHYL aro solution for 15 seconds.

Then gently swirl the solution and microwave for another 15 seconds. Be careful when swirling the ARO 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 ARO solution in a 45 degree Celsius water bath during the next steps to prevent the agro solution from solidifying prematurely.

Next, transfer 12 milliliters of warmed media using the Prewarm pipettes into a sterile 50 milliliter conical tube. Immediately add three milliliters of the 3%aros solution and gently invert the conical tube to mix the aros 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 degree 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 agra shell do not solidify before addition to each well to ensure that the seltz are mixed in the media before adding the liquid agri gel. Additionally, the pipes are prewarm beforehand to avoid the solutions from solidifying while handling. To prepare the cell containing layer first trypsin eyes MCF 10 DCIS cells and dilute them to a cell concentration of 40, 000 cells per milliliter, then take eight milliliters of media using prewarm pipettes and transfer into a sterile 50 milliliter conical tube.

Immediately add two milliliters of 3%aros to the conical tube and gently invert to mix the aros with the media. Avoid forming any bubbles. Next, take two milliliters of the cells and treat them with two micromolar BB chloramine in DMSO or DMSO alone as the control following treatment, mix the cells with the 0.6%aros in a one-to-one dilution to make a final concentration of one micromolar BB chloramine.

Then take one milliliter of the cell aros 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%two hydroxyethyl aro solution as before and equilibrating the agro solution bottle in a 45 degree Celsius water bath mix one milliliter of 3%agro solution with nine milliliters of warm media into a 50 milliliter conical tube and gently invert to mix the aros with the media. Avoid forming air bubbles. After treating the mixture with BB chloramine 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. Bader, repeat this feeding procedure weekly by overlaying one milliliter of 0.3%agros medium treatment solution onto the existing feeder layer to replenish the cells with new media until colony formation is observed.

After two and a half weeks of cell growth in the soft agar, the colony number in each well is counted. Using a light microscope to facilitate quantification, print a grid onto a transparency and attach the grid to the six well plate to help locate where the cells are during counting. Colony size is quantified by the diameter of each colony and will vary predefine a reference colony size to determine which colonies will be scored.

For example, here, colony sizes of 70 micrometers or larger are included in the data analysis. After counting seal the six well culture plate with paraform to prevent the gels from drying out. Store the samples at four degrees Celsius to prevent further colony formation and for future counting.

The results demonstrate that BB chloramine significantly inhibits the formation of MC 10 DCIS cell derived colonies in the presence of a pad inhibitor. There was a reduction in both colony formation and colony size when compared to the DMSO control. The size of colonies for BB chloramine treated MCF 10 DCIS cells was predominantly within the range of 20 to 100 micrometers.

While the size of colonies for the DMSO control exhibited a greater range of 70 to 150 micrometers after two and a half weeks of growth colonies larger than 70 micrometers were counted and analyzed. There was an average of about 3, 500 colonies in the DMSO control, whereas only approximately 2000 colonies were seen in the BB chloramine treated group. After two and a half weeks of soft agriculture, this represents a 44%decrease in the average colony formation in the presence of one micromolar BB Chloramine indicating a significant tumorogenic inhibition of breast cancer cells by the pad inhibitor.

While attempting this procedure, it is important to keep in mind to prewarm all the reagents and equipments beforehand to avoid the solutions from solidifying when handling, and thanks for watching.