I have recently began culturing a melanoma cell line in hanging drop plates to form spheroids with the intention of harvesting the spheres into the bottom of a 96-well round bottom plate and covering them in matrigel to conduct a 3D invasion assay.
I am able to readily form spheroids in the drop plates however after harvesting I've found that all of the spheres actually float in standard media. I am unable to spin the spheroids down and this is preventing me from capturing the sphere in matrigel at the center of the round bottom plate.
Does anyone have any advice for making my spheroids more dense so they don't float? Is this a common problem?
how long do leave them to sink down. usually it takes a few minutes for them to sink down. it also depends how many cells you use for your spheroids.
Alternatively you could change the protocol to the liquid overlay method:
Smalley KS, Lioni M, Noma K, Haass NK, Herlyn M (2008; Epub 2007 Dec 19). In vitro three-dimensional tumor microenvironment models for anticancer drug discovery. Expert Opin Drug Discov 3: 1-10
Beaumont KA, Anfosso A, Ahmed F, Weninger W, Haass NK (2015) Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids. J Vis Exp 106: e53486, pp. 1-9, doi:10.3791/53486
We also have an accepted methods paper with the detailed protocol. I can send that to you if you wish.
These spheroids never sink. I've left them for hours even days in the incubator. I've tried seeding 2000, 5000, and 10000 cells. The spheres are different sizes but they all respond the same.
Nikolas-
Thank you suggesting these other protcols. If you'd like please send me the methods paper and I will take a look.
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