Check out the attached paper

Dear Pushkar!

Here is the essay: 

The Soft Agar Colony Formation Assay

URL: http://www.jove.com/video/51998

DOI: doi:10.3791/51998

Keywords: Cellular Biology, Issue 92, Wnt, Frizzled, Soft Agar Assay, Colony Formation Assay, tumour suppressor, lung cancer

Abstract

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface and is a hallmark of carcinogenesis.

The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the

most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line,

CMT167, to demonstrate a tumour suppressive effects of two members of the Wnt signalling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent

overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumour growth in a murine lung carcinoma model.

Video Link

The video component of this article can be found at http://www.jove.com/video/51998/

Introduction

The soft agar colony formation assay is a technique widely used to evaluate cellular transformation in vitro. Historically, another assay, the

clonogenic assay, described by Puck et al. in 1956 was used to evaluate the ability of cells to form colonies. In this technique, cells were

dispersed onto a culture plate and grown in the presence of 'feeder' cells or conditioned medium to provide necessary growth factors. The

limitation of this technique was that it only provided information regarding colony formation. Normal cells are prevented from anchorage-independent growth, due to a particular type of apoptotic death, called anoikis. However, transformed cells have the capability to grow and divide without binding to a substrate. To capitalize on this concept, researchers developed the soft agar colony formation assay. The soft agar colony formation assay has since been modified, in more recent years, to address specific needs. One variation involves incorporation of fluorometric dye to allow for high-throughput colony counting. Another variation involves the use of specialized agar solution to allow for retrieval of viable cells after colony formation when protein or DNA samples are needed.

In the traditional soft agar colony formation assay, cells are grown in a layer of soft agar mixed with cell culture medium that rests on another

layer of soft agar, also mixed with cell culture medium, but containing a higher concentration of agar. This prevents cells from adhering to the

culture plate, yet allows transformed cells to form visible colonies. The rationale behind this technique is that normal cells depend on the cell to extracellular matrix contact to be able to grow and divide. Conversely, transformed cells have the ability to grow and divide irrespective of their surrounding environment. Therefore, cells able to form colonies in an anchorage-independent manner were considered to be transformed and

carcinogenic. The overall goal of this method is to measure this capability in cells in a semi-quantitative and stringent manner.

The Wnt signalling pathway is critical in embryogenesis and often de-regulated in tumorigenesis3-6. There are multiple pathways associated

with Wnt signalling. The canonical pathway involves Wnt signalling and regulation of downstream gene transcription through its effects on

the transcriptional coactivator beta-catenin. Wnts also signal through several non-canonical pathways, for example, the planar cell polarity

pathway, which regulates elements involved in cytoskeletal structure

7, and the Wnt-calcium pathway, which regulates the release of calcium from the endoplasmic reticulum. Wnt ligands exert their activity through binding Frizzled receptors. Although several Wnts have been shown to be upregulated in lung cancer, Wnt7a has been shown to be down-regulated in non-small cell lung cancer through promoter methylation. Wnt7a binds Fzd9 and acts as a tumour suppressor through a non-canonical pathway. Restoration of Wnt-7a and Fzd-9 inhibits the growth of non-small cell lung cancer cells. The effects of Wnt7a/Fzd9 are mediated through the activation of ERK-5, which in turn, activates peroxisome proliferator-activated receptor γ (PPARγ). Here, we show that overexpression of Wnt7a and Fzd9 results in the suppression of anchorage-independent

The growth of a murine lung carcinoma cell line. Murine CMT167 cells were derived from a lung carcinoma in C57BL/scarf mice13 and were stably

transfected with Wnt7A and Fzd9. Overexpression of Wnt7A and Fzd9 were confirmed by quantitative-PCR (Q-PCR) and the functionality of

Wnt7A and Fzd9 overexpression were confirmed through downstream activation of PPARγ.

Protocol

Preparation of Materials and Reagents

1. Label each well of a tissue culture treated 6-well plate appropriately for each cell line or condition being investigated.

2. Prepare 2x cell culture medium by dissolving 1 g of powder medium and 0.2 g of sodium bicarbonate in de-ionized water to a final volume of

50 ml.

3. Pass this medium through a 0.2 μm filter to sterilize.

4. Add additional components needed for the normal culture of the cell line of interest. For example, grow CMT 167 cell line in RPMI 1640 medium

supplemented with 10% FBS and 1% penicillin/streptomycin solution. Warm medium to 37 °C in hot water bath prior to use.

5. Prepare 1x cell culture medium separately as you would for normal cell culture of the cell line of interest.

6. Prepare 1% noble agar by adding 1 g of noble agar to 100 ml of deionized water.

NOTE: Noble agar will not dissolve completely with agitation alone.

7. Prepare 0.6% noble agar by adding 0.6 g of noble agar to 100 ml of de-ionized water. Both agar solutions can be made in 100 ml glass

bottles with closable lids for long-term storage.

8. Autoclave the noble agar mixtures to sterilize. These mixtures can be made in advance and stored at 4 °C but should be heated again at the

time of the experiment until agar has completely dissolved.

9. Prepare nitroblue tetrazolium chloride solution by making a 1 mg/ml stock solution in 1x PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24

g KH2PO4 in H2O to the final volume of 1,000 ml). This will be used at the end of the experiment to stain the colonies.

2. Plating of Bottom Layer of Agar

1. Loosen the cap on the bottle of 1% noble agar and microwave for about 1-2 min. While heating in a microwave, monitor the solution closely to

avoid boiling over. Continue heating, while mixing intermittently, until agar is completely dissolved and the solution is clear.

NOTE: Use heat-resistant gloves to handle flask after heating. Failing to do so may cause the burn or serious injury.

2. Place melted agar solution and pre-warmed 2x culture medium in an ice bucket filled with hot tap water (42 °C). Also, place a 50 ml conical

tube in a tube holder in the ice bucket with hot water. Transfer bucket to cell culture hood for subsequent steps.

3. For the bottom layer of agar, you will need 1.5 ml of a mix of agar and medium per well of a 6-well plate.

4. To ensure an adequate amount of the mixture, prepare a total of 12 ml for each 6-well plate.

5. Start by adding 6 ml of culture medium to the 50 ml conical tube and then 6 ml of the 1% noble agar solution.

6. Invert the conical tube several times to mix. Working at a brisk pace will prevent premature hardening of the soft agar.

7. Draw up approximately 5.5 ml of mixture into a 5 ml serological pipette.

8. Allow the air bubbles to rise to top of pipette column before depositing 1.5 ml of this mixture into each well. Use caution to avoid deposition of

any air bubbles into the plate wells.

9. Cover the plates and allow agar mixture to solidify at room temperature, in cell culture hood, for 30 min.

3. Plating the Upper Layer of Agar Containing Cells

1. Once the lower layer of agar has solidified, begin preparation of the upper layer.

2. First, harvest cells by trypsinization and dilute them 1:5 in culture medium (e.g. for 1 ml of trypsin, add 4 ml of medium) into a 15 ml conical

tube.

3. Count cells and calculate the number of cells needed per well to prepare a final cell suspension at this time. This number will vary depending

on cell type. Use 5,000 cells/well as a starting point and adjust as needed. For this cell number, you would prepare a cell suspension of 6,667

cells/ml (i.e. each well will receive 0.75 ml of this suspension and 0.75 ml of agar for a total volume of 1.5 ml; the concentration of cells will

also be diluted 1:2 for a final total cell count of 5,000).

4. The volume of cell suspension needed per well of a 6-well plate will again be 1.5 ml. Prepare additional cell suspension totaling 12 ml per 6-

well plate.

5. Melt 0.6% agar solution in a microwave as above and place into ice bucket containing hot water along with a 50 ml conical tube in a tube

holder and the final cell suspension from Step 3.3.

6. Transfer the ice bucket with melted 0.6% agar to cell culture hood for subsequent steps.

7. Mix 0.6% agar and cell suspension in a 1:1 ratio, preparing a total volume of 12 ml per 6-well plate. 1.5 ml will be required per well but extra

should be made as above.

8. Pipette 6 ml of cell suspension into the 50 ml conical tube.

9. Then, add 6 ml of 0.6% agar to the tube.

NOTE: The temperature of this mixture must be kept around 42 °C to avoid premature hardening and to maximize cell survival.

10. Working quickly, pipette this mixture 2-3x to distribute cells, then draw up 5.5 ml of mixture into a 5 ml serological pipette.

11. Allow any air bubbles to rise to top of pipette column before depositing 1.5 ml of this mixture into each well. Use caution to avoid deposition of

any air bubbles into the plate wells.

12. Allow cell/agar mixture to solidify at room temperature, in cell culture hood, for 30 min before placing into a 37 °C humidified cell culture

incubator.

13. The time required for adequate colony formation varies for each cell line, typically around 21 days.

14. A layer of growth medium should be maintained over the upper layer of agar to prevent desiccation. 100 μl of medium added twice weekly is

sufficient for this purpose.

4. Staining the Plates and Counting Colonies

1. Stain cells by adding 200 μl of nitroblue tetrazolium chloride solution per well and incubating plates overnight at 37 °C.

2. Once colonies are stained, take photographs of wells using an image and count colonies using image analysis software 

In addition here is the complete article!

Sincerely yours!

Dr. Bratko Filipič

Email: [email protected]

Thank you Dr. Bratko Filipic!! I am going to try this and let you know soon :)

Check out the attached paper

Thank you Dr. Philip. This paper is fantastic!

Dear Dr Philip, 

A lots of thanks from my side too because it is excellent article for Clonogenic assay of cells for freshers. who wants to do work in the field of cell biology, molecular biology and oncology etc.

Hi everyone,

I am studying renal cell carcinoma and would like to do soft agar assays. However, I would like to collect their proteins at the end of the experiment. I am having difficulty finding a protocol that covers 'specialized agar solution to allow for retrieval of viable cells". Any experience or suggestion would be greatly appreciated.

Kindest regards,

Canh-Vinh

Hi Canh-Vinh

You should be able to collect the cells from the agarose by adding medium and resuspending the mixture followed by several centrifugation steps.