28: Soft agar colony formation assay
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
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
- 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. ↵
- 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. ↵
- 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. ↵