I was performing wound healing assays with PC-3 cells (cultured in RPMI+10%FBS; 500,000 cells/well in 6 well plate). I seeded them for 24 hours, then applied the scratch with a 100uL pipette tip. they looked like the first picture under the microscope. seems pretty normal, i think
Yet 16 hours later, they looked like the second and third pictures
I'm wonder what does that say about the cells' condition, and whether I should do something to prevent that from happening next time...
THANX!!
Can you check if the microscope incubator is working fine? Sometimes it may happen due to insufficient temperature / CO2. A simple check would be to keep another plate in the CC incubator and check after a fixed time. Something similar happened to me, so instead of constant imaging, I imaged at intervals of 3 hours at multiple similar regions.
My first reaction is to say the cells have outgrown their space and nutrition. They look crowded.
You should definitely seed less amount of cells. For wound healing experiments add anti-migrating agents.
Good luck!
The images after 16h look fine to me. The thread like structures could be because of migration of the cells into the wound. However, the cells density is higher after 16h indicating that the cells are overcrowding. For migration assay, you should use mitomycin C to suppress cell proliferation to make sure that the wound healing is because of migration and not the cell proliferation.
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