Because spheres of cancer stem cells are non-adherent, it is hard to separate them from dead cells in the supernatant
Its possible. See my paper
https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis
Sphere forming cells are adherent by the way.
Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...
The type of experiment being done, or the source/number of dead cells, is not clear. However, there are a few simple things one could try to remove CSC spheres from dead cells in culture:
1. Gravity sedimentation: Suspend spheres in tissue culture medium in a conical centrifuge tube and let spheres settle to the bottom. Carefully remove most of the supernatant and transfer the spheres collected in the bottom of the tube to a new culture plate. Volume of the medium, and time required for sedimentation of the spheres, would need to be optimized for best recovery.
2. Cherry-picking spheres: If the number of spheres is small, use a large bore pipet to transfer the spheres to a new cell culture plate.
3. Pre-plating step: Briefly plate spheres on a culture plate and allow to adhere. Wash with medium or PBS to remove dead cells. Sphere cells would be ready for culture – either on plastic or in suspension. The minimum time required for sphere attachment would need to be optimized. Hopefully the dead cells would not adhere to the plate during that time period. Selective adherence could also be optimized by using different types of culture plates. For example, epithelial cells preferentially adhere to Primaria plates while fibroblasts preferentially adhere to bacterial plates. One could also try ECM-coated verses uncoated culture plates.
4. CSCs could be isolated by MACS prior to culture.
are cancer spheres much different than neuro spheres (stem cells)?
I thought not. In which case I can suggest a low G centrifugation, for instance 200 g for 7 min to separate spheres from dead cells and debris...
We just use gravity sedimentation. It only takes less than one minute for them to settle to the bottom of a conical tube, then carefully pipette out the remaining media. You can do several washes like this to get rid of most dead cells. Don't use a vacuum aspirator to remove the media or you will loose all your spheres!!
All opinions it's good
also , you can read the reference in the attachment , will give you all the answers
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