Invasion assay: analysis question: round cells - (Mar/18/2015 )

Hello everyone,

I am studying invasion behaviour of TOV112D ovarian cancer cell line. We are using a method which involves 2 stains so the invaded cells are red and the non invaded cells are blue, which makes it easier to distinguish and count.

This works quite well actually but I often see differences in the shapes of cells: the blue (non invaded ones) are often small and round but the red ones are always looking nicely shaped (like they grow in the culture flask). There are also many blue ones which have nice shapes, and the round ones do not look dead but just round.

I am not sure whether I should include these round cells in my analysis or just not count them..? And, more importantly, does anyone have a clue what the cause could be? Why do the cells look like that (picture attached, 10x magnification)?

My protocol (briefly) is

1) Thaw inserts (30 mins)   FAL554483 Falcon Biocat

2) equilibrate with serum free medium (2h)

3) add trypsinized cells (30.000) in  medium w 1% FBS to upper chamber and medium w 10% FBS to lower chamber

4) incubate 48h

5) fix, stain, analyze

I am happy to go into more detail if anyone has a suspicion. Thanks a lot!

Do you wash after you stain them? The round and (dark) blue cells look dead to me ... but then again, I'm not familiar with this cell line at all. If all the cells are supposed to adhere, my suspicion is that the round cells are dead as they have not detached. Is your stain selective for live/dead cells? My thought is that you should exclude them from your analysis.

Thank you SusieQ! Yes I do wash after staining. I am also tending to exclude these cells as they look dead to me too. However I find it hard to draw a clear line because there are all kinds of in between morphologies...Our staining is not selective for live/dead cells. Thanks again!