I am interested in understanding the role of an enzyme in cancer stem cell function
and I over expressed this enzyme (stable transfection). My flow date ie., side population assays show good increase in side populated cells, whereas colony formation and sphere formation show opposite results. I am having a very hard time understanding this. The only thought I have is that this enzyme cleaves the matrix molecules so I am suspecting this may be it. Could anyone help me out from this problem please?
Interesting. First make sure you are doing right gating for side population. See the paper in following link for details. I would recommend verapamil usage to confirm side population you get (it should reverse side-population).
http://www.cell.com/cell-stem-cell/abstract/S1934-5909(11)00008-7
Second, there are different types of sphere formation assays. One is soft-agar colony formation and the other is blebbishield mediated sphere formation after apoptosis.
https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis
Both are suitable for cancer stem cell studies. Blebbishield are shown to form from low Hoechst-33342 stained cells (equivalent to side-population).
For blebbishields, you might use any therapeutic that can induce apoptosis (typically 24hrs) in your cell type and collect the apoptotic cells gently, pellet it at low speed (1200 rpm), re-suspend with wide mouth pipette (gently), and plate it in fresh medium and observe after 10-20 hours for sphere formation.
Good luck..!!
J.
Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...
What cells are you working with? Side population analysis is a very sensitive technique. I hope you are using proper controls: you may like to use Verapamil or better KO143 to inhibit SP that will help you set up a SP-gate.
best wishes,
Farid
hello, what cell type are you using? SP may not always correlate with other stem cell properties (see e.g http://www.ncbi.nlm.nih.gov/pubmed/21425408 for glioma cells). you may want to check for the expression of the various (imperfect...) stem cell markers in your transfected cells or implant the cells into immunodefficient animals to see the effect on tumor formation. in effect, you want to know what the enzyme does with the tumorigenicity of your cells and not with the ability to exclude the hoechst dye.., right? good luck, petr
There are basically two majorly used dyes (Hoechst or DyeCycle Violet) that can help you in determining the Side Population in any cancerous cells. As you may know that the SP assay works on the principle of the ability of the cells to efflux out the drug. Generally Verapamil is used as a drug control in combination to either of Hoechst or DCV.
The reason for using this verapamil control is so that it can help you determine the correct gating of your side population cells.
The below two articles should help you better understand in depth about the side population assay.
http://www.sciencedirect.com/science/article/pii/S0304383508002231#
http://www.sciencedirect.com/science/article/pii/S0014482706003636
Hope it helps.
Good luck.
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