Higher temperatures may harm the microorganisms. 1.8% agarose . This type of soft agar assay can give insight into two important properties of a subset of cancer cells. Bottom layer is 2.5 ml of 0.5% Agar Noble (in DMEM) per well and top agar is 1 ml of 0.33% Agar Noble per well (with 25,000 to 100,000 cells in MEGM). The noble agar assay is based on the principle that the cancer cells are anchorage-independent and do not show contact inhibition. However, this method is lengthy (3-4 weeks incubation), laborious (counting colonies) and inconsistent (due to subjective counting). Would suggest you try chilling the bottom layer at 4oC before plating the top layer. Use autoclaved 125 mL screwtop bottle b. The most primitive cancer cells are expected to have the highest tumorigenic efficiency. The main difference between Plaque Assay and Colony-Counting is that, to count bacteria, we spread about 100 - 400 bacteria over the surface of the agar and incubate the plate. The molten agar should be maintained in a water bath at a temperature not more than 45°C. If problems persists maybe try a slight increase in agar concentration of the bottom layer (0.7%? ½ 2xDMEM 2ml. Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. Sterile, solidified agar can be re-melted only once. Soft Agar Assay Protocol 1. Many protocol improvements were made to the soft agar version of the MLA in the 1980s but in order to overcome the problem of agar quality, particularly the effect on recovery of small colony mutants, Cole et al.
I left the bottom agar layer at room temp for an hour and then put the plates in 37C incubator overnight before putting top layer. The Soft Agar Assay for Colony Formation is an anchorage independent growth assay in soft agar, which is considered the most stringent assay for detecting malignant transformation of cells. 1/3 1.8% agar 1.3ml. For the first time, I am doing it in 6-well plate format (by titrating everything down based on the surface area differences between different plates). Things to prepare: 2xDMEM. , developed the microwell version of the assay growing the cells in liquid medium in 96-well plates. Preparation of Base Agar: a. Dissolve 1% agarose (Difco Agar Noble) in sterile H20, cool to 42°C in water bath. For this assay, cells (pretreated with carcinogens or carcinogen inhibitors) are cultured with appropriate controls in soft agar medium for 21-28 days. Traditionally, the soft agar colony formation assay is a common method to monitor anchorage-independent growth, which measures proliferation in a semisolid culture media after 3-4 weeks by manual counting of colonies. The molten agar should not be kept in the bath for more than three hours. To count phage, we spread 100 - 400 phage particles mixed with a very large number of host bacterial cells over the surface of the agar and incubate the plate. 8. This type of soft agar assay can give insight into two important properties of a subset of cancer cells.
Problem with soft agar assay - (Jan/29/2012 ) I've done soft agar assay for several times in past in various formats. Read our 11 Pour Plate Method Best Practices post for more tips. Procedure: Make bottom agar (0.6%): Melt the 1.8% agarose completely in microwave, place it in 42(C to keep it from solidifying. i. These are breast cancer cells. So, probably they were set well-but may be putting in incubator overnight before plating top layer was a mistake?Changing the format may increase the time the bottom agar needs to FULLY set. If noble agar solution is added to cold (or even room temperature) DMEM, solidification will begin too soon and you will not be able to proceed with the assay.Step 4b: Be sure to immediately pour this solution onto the plates, otherwise the solution will solidify in the 10cc tube.The noble agar assay is based on the principle that the cancer cells are anchorage-independent and do not show contact inhibition. Construct bottom agar mix as following. The colony is defined to consist of at least 50 cells. Secondly, the self-renewal properties of cancer cells can be assessed based on the types of progeny produced in noble agar.Step 5b: Be sure to immediately pour this solution onto the plates, otherwise the solution will solidify in the 10cc tube. Standard soft agar assays are usually performed in 100-mm or 60 mm This is based on the ability of cells to grow unattached and to remain suspended in agar. ). Warm 2X DMEM w/20% FBS and antibiotics to 42° in water bath. Soft agar assay protocol.
Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation Asaf Rotem a, Andreas Janzera,1, Benjamin Izar b,c, Zhe Ji , John G. Doenchc, Levi A. Garraway , and Kevin Struhla,2 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; bDepartment of Medical Oncology,