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