Hi, I wanna ask about invadopodia formation assay. I'm using MDA-MB231 breast cancer cell line for these assay but I didn't get invadopodia. An addition I use RPMI media, gelatin 0.5mg/ml, phalloidin dye, and incubation 3h for invading, so is there any suggestion about this condition ?? thank you in advance.
Indavopodia formation takes some time. Which protocol do you use? In my case (I also use MB231 cells) it takes about 5 days to see invadopodias. At first I grow spheroids from single cells in the mix of collagen/fibronectin/matrigel, and I use the medium containing 20% FBS. Then invadopodia form very nicely, and after about 7 days it is possible even to see cell migration through them.
thank you for replay, actually a used RPMI media containing 10% PBS. please find attach the file. thank you in advance
In our experience, 231 cells do not form a lot of invadopodia nor degrade very much ECM. We have also noticed that they are quite sensitive to the ECM used in the invadopodia assay as well as the amount of cross-linking. In addition, recent work I saw at the last ASCB meeting found that hypoxia actually induces significantly greater invadopodia activity by these cells. You also may want to look in the literature since many labs use higher amounts of FBS plus supplements (like EGF) that promote invadopodia formation.
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