The overall goal of this procedure is to quantify cellular anchorage independent growth. This is accomplished by first plating a layer of agar onto the cell culture plate and allowing it to harden. The second step is to plate a mixture of cells in agar over the top of the previous agar layer.
Next, the cells are allowed to form colonies over a period of a few weeks. The final step is staining the colonies for visualization and quantification. Ultimately, the soft agar colony formation assay is used to show Anchorage independent growth as an indication of cellular transformation.
The main advantage of this technique over other methods, such as the Clon genic assay, is that the soft agar colony formation assay requires cells to form colonies in an Anchorage independent manner. Begin the procedure by labeling each well of a tissue culture treated. Six well plate appropriately for each cell line or condition being investigated.
Next, prepare two Xcel culture medium by dissolving one gram of powder medium and 0.2 grams of sodium bicarbonate in the deionized water to a final volume of 50 milliliters. Pass this medium through a 0.2 micron filter. Add additional components needed for normal culture of the cell line of interest.
For example, grow CMT 1 67 cell line in RPMI 1640 medium supplemented with 10%FBS and 1%penicillin streptomycin solution. Warm medium to 37 degrees Celsius in hot water bath prior to use. Now prepare one Xcel culture medium.
Then prepare 1%noble agar by adding one gram of noble agar to 100 milliliters of deionized water. After that prepare 0.6%noble agar by adding 0.6 grams of noble agar to 100 milliliters of deionized water. Next autoclave the noble agar mixtures.
Subsequently make a one milligram per milliliter nitro blue tetra oleum chloride stock solution in one XPBS for the purpose of staining the colonies. In this procedure, microwave the bottle of 1%noble agar for about one to two minutes while heating in a microwave. Monitor the solution closely to avoid boiling over.
Then continue heating and mixing intermittently until the agar is completely dissolved and the solution is clear. After that, place the melted agar solution and the Prewarm two x culture medium in an ice bucket filled with 42 degrees Celsius. Tap water.
In addition, place a 50 milliliter conical tube in a tube holder in the ice bucket with hot water. Next, transfer the bucket to the cell culture hood. Then add six milliliters of culture medium and six milliliters of the 1%noble agar solution to the 50 milliliter conical tube.
Pipette up and down several times to mix. Working at a brisk pace will prevent premature hardening of the soft agar. Afterward, draw up approximately 5.5 milliliter of mixture with a five milliliter serological pipette.
Allow the air bubbles to rise to the top of the pipette column. Then deposit 1.5 milliliters of the mixture into each well and avoid the deposition of any air bubbles. Cover the plate and allow the agar mixture to solidify in the cell culture hood at room temperature for 30 minutes.
First, harvest cells by trypsin and dilute them one to five in culture, medium into a 15 milliliter conical tube. Count the cells and calculate the number of cells needed per well to prepare a final cell suspension at this time. Next melt 0.6%agar solution in a microwave.
Place it in an ice bucket containing hot water along with a 50 milliliter conical tube in a tube holder and the final cell suspension. Then transfer the ice bucket with melted 0.6%agar to the cell culture hood for subsequent steps. To prepare a total volume of 12 milliliters of cell suspension for a six well plate first pipette six milliliters of cell suspension into the 50 milliliter conical tube.
Then add six milliliters of 0.6%agar to the tube. Keep the mixture at 42 degrees Celsius to avoid pre premature hardening and to maximize cell survival. Afterward, pipette the mixture quickly, two to three times to distribute the cells.
Draw 5.5 milliliters of mixture with a five milliliter serological pipette and allow any air bubbles to rise to the top of the pipette column. Then deposit 1.5 milliliters of the mixture into each. Well allow the cell agar mixture to solidify at room temperature in the cell culture hood for 30 minutes before placing it in a 37 degrees Celsius humidified cell culture incubator.
Add 100 microliters of growth medium over the upper layer of agar twice a week to prevent desiccation and allow around 21 days for adequate colony formation. To stain the cells, add 200 microliters of nitro blue tetra oleum chloride solution to each well and incubate the plate overnight at 37 degrees Celsius. Once the colonies are stained, take photographs of the wells using the imager and count the colonies using image analysis software.
Shown here is the soft agar colony formation assay of vector expressing CMT 1 67 LNCX LPC X cells and CMT 1 67 LL went seven a frizzled nine over expressing cells. The vector expressing CMT 1 67 L and CXL PC X cells and CMT 1 67 LL went seven a f frizzled. Nine over expressing cells were plated in a soft agar colony formation assay CMT 1 67 LL went seven frizzled nine cells showed a marked decrease in colony formation After its development.
This technique paved the way for researchers in the field of cancer research to explore cellular transformation in vitro. After watching this video, you should have a good understanding of how to assess cells for Anchorage independent growth.
