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28: Soft agar colony formation assay

  • Page ID
    135780
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    Summary

    The soft agar colony formation assay is a test to determine whether cells are anchorage-independent. Anchorage independence is a characteristic of cells that have undergone malignant transformation.

    Also known as

    Test of anchorage independence

    Samples needed 

    Cells capable of growing in tissue culture

    Method

    Normal, non-transformed cells only grow attached to a solid substrate. This characteristic is called anchorage dependence. A distinctive feature of transformed cells is their ability to grow without that solid substrate; they are anchorage independent. To perform the test for anchorage independence, cells are plated in agar, a soft, jelly-like substance, and supplied with the necessary nutrients and growth factors. After 2-3 weeks, the plates are stained to aid in colony identification, then photographed, and the colonies are counted.[1]

    Controls

    The soft agar colony formation assay is nearly always used to compare anchorage-independence of cells +/- a treatment or intervention. Therefore, the usual mock treatment controls apply.

    Interpretation

     

    Colony formation assay. Image description available.
    Figure 1. A soft agar colony formation assay on H1299 cells with either wild type METTL3 protein or METTL3 with a mutation that turns four key lysine residues to arginine residues to prevent SUMOylation. Relevant section of caption for published figure reads: “(A) The mutation of 4KR in METTL3 reduces the soft-agar colony formation of H1299 cells. H1299-shMETTL3 cells stably re-expressed METTL3-WT or METTL3-4KR cell lines were seeded in 2 ml of medium containing 10% FBS with 0.35% soft agarose at 1000 per well and layered onto 0.6% solidified agarose. The photographs were taken 14 days after seeding, and the number of colonies were counted and analysed. Each value represents the mean±s.e.m. of three independent experiments with triplicates.” “Figure 5” by Yuzhang Du, Guofang Hou, Hailong Zhang, Jinzhuo Dou, Jianfeng He, Yanming Guo, Lian Li, Ran Chen, Yanli Wang, Rong Deng, Jian Huang, Bin Jiang, Ming Xu, Jinke Cheng, Guo-Qiang Chen, Xian Zhao, Jianxiu Yu[2]. [Image description]

    This figure shows the reader that in H1299 non-small cell lung cancer(NSCLC) cells, the SUMOylation of METTL3 is important for the process of transformation. This is evident because if METTL3 cannot be SUMOylated (4KR mutatnt), significantly fewer colonies are able to form in soft agar.


    Image Descriptions

    Figure 1 image description:

    Left: Six cell culture plates containing purple-stained soft agar. In each plate, there are some darker purple spots showing cell colonies. There are more colonies in the three wild type plates compared to the mutant plates. Right: A column graph quantifying colony growth. The wild type condition shows about 140 colonies per 1000 cells, whereas the mutant condition shows about 80. The difference is significant. ↵

    Thumbnail

    "Figure 2"↗ by Wang et al. is licensed under CC BY-NC 3.0↗. See reference 3.

    Description: Colony formation assay plates.

    Author

    Katherine Mattaini, Tufts University


    1. Borowicz, S., M. Van Scoyk, S. Avasarala, K. Karuppusamy Rathinam Manoj, J. Tauler, K. Bikkavilli Rama, and R. A. Winn. 2014. The soft agar colony formation assay. JoVE :e51998. 
    2. Du, Y., G. Hou, H. Zhang, J. Dou, J. He, Y. Guo, L. Li, R. Chen, Y. Wang, R. Deng, J. Huang, B. Jiang, M. Xu, J. Cheng, G. Chen, X. Zhao, and J. Yu. 2018. SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function. Nucleic Acids Research 46:5195-5208. 
    3. Wang, H., Z. Shao, Z. Xu, B. Ye, M. Li, Q. Zheng, X. Ma, and P. Shi. 2022. Antiproliferative and apoptotic activity of gemcitabine-lauric acid conjugate on human bladder cancer cells. Iranian Journal of Basic Medical Sciences 25:536–542. 

    This page titled 28: Soft agar colony formation assay is shared under a CC BY-SA 4.0 license and was authored, remixed, and/or curated by Katherine Mattaini.

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