we observed lower clonogenocity in our transfected cells ( clonogenic assay was performed in adherent condition seeded at low density) but cell cycle was not altered. how we can explain this?
Paracrine signaling can influence cell growth. Seeding cells more than 50% increases its growth rate than lower one.
Thank you for your suggestion you mean that the self-renewal property of cancer cells was affected!
I have several questions and suggestions:
1: How long is the time between transfection and harvesting cells for cell cycle analysis? If it is too short in comparison to that of the clonogenic assay, you can also harvest samples at later timepoints to see if there is any difference. May be the effect takes a longer time to be more obvious.
2: After transfection, did you see any growth inhibition if you are not seeding at low density? You may try culture and propagate transfected cells together with control cells as usual to see if proliferation is inhibited.
3: By "cell cycle was not altered", do you get this answer simply from looking at cell cycle profile of asynchronous cells? If yes, doing block and release experiments with reagents (e.g. mimosine, thymidine, hydroxyurea, nocodazole, RO3306, etc.) that block cells at different phases would give more accurate results. Sometimes simply looking at the cell cycle profile of asynchronous cells may not give you obvious results.
Comparing with appropriate controls like seeding same number of cells transfected with control vector can conclude relative clonogenic potential of our transfected cells.
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