Methylene Blue Staining to Assess Metastatic Colony Formation In Vitro: A Staining Procedure to Assess Metastasis by Quantifying Harvested Cancer Cells From Cancer Mouse Model

Bring the volume of the tube up to 10 milliliters with 1X HBSS. Then, pour the contents over a 70-micrometer cell strainer into the 50-milliliter conical tube. Use the plunger of a 1-milliliter syringe to gently grind the sample through the strainer.

Centrifuge the tube for 5 minutes at 350 times g, and discard the supernatant. Then wash the pellet twice with 10 milliliters of 1X HBSS. Resuspend the pellet in 10 milliliters of 60 micromolars 6-Thioguanine complete culture media, and plate the samples in the 10-centimeter cell culture plates using a dilution scheme, if desired.

Incubate the plates at 37 degrees Celsius and 5% carbon dioxide for 5 days. Pour culture media off of the plates into the appropriate waste container. To fix the cells, add 5 milliliters of undiluted methanol to each plate, and incubate for 5 minutes at room temperature, making sure to swirl the methanol so that it covers the entire plate.

Pour the methanol off the plates into the appropriate waste container, then, rinse each plate with 5 milliliters of distilled water. Add 5 milliliters of 0.03% methylene blue per plate, and incubate it for 5 minutes at room temperature, making sure to swirl the methylene blue solution so that it covers the entire plate. Pour the methylene blue into the appropriate waste container, and rinse each plate again with 5 milliliters of distilled water.

Turn the plate upside down and blot it against a paper towel to remove excess liquid. Place the plate on its lid, and let it air dry overnight. Metastatic colonies will be blue. Once the plates have dried, they can be stored at room temperature indefinitely.