Problems with invasion assay - (Dec/03/2014 )

Hello,

I am sorry for the long message but I am frustrated because my invasion assays do not work. I want to test the invasive ability of HepG2, Huh7 and Hep3B cells. 

I've tested 10,000 , 20,000, 30,000 and  50,000 cells for 12h, 24h, 30h and 48h, but I saw no or very few migrating cells depending on the cell line (migratory cells were present in 20,000 seeded HepG2 cells and 30,000 Huh7 cells. Nevertheless, duplicate experiments even for 48h were totally unsuccessful! ).  

 Besides that, I cannot see the nucleus in HepG2 cells after staining  thus, I am uncertain on whether what I see are cells (stained "stuff" seem to float after the final wash and then settle on the mebrane after drying. They look more like cell debris or "dirt" than cells).  Can cells change in morphology after invasion? The cells that I see are small in size, round or unevenly shaped.

I do not think, that I am loosing cells during staining and washing because I check the membranes under the microscope right after removing the matrix.

 I do not fix cells in methanol prior staining because my stainning solution contains 50% methanol. Is this ok? 

The kit's manufacturer  suggests to use 150,000-300,000 cells per well (24-well plate) ,which I think are too many and I am afraid that my cells will clump (especially HepG2).

In the insert I use DMEM/1% FBS. I have tried pre-treating my cells for 24h with 1% FBS medium but all my cells died.  So I trypsinize cells as usual and then wash them x2 in 1% FBS/DMEM. At the bottom chamber I use 20% FBS/DMEM. Inhibitory agents are added in both the insert and the bottom chamber. Am I doing something wrong here?

 I am planning to seed 150,000 cells/well in serum-free medium and 10% FBS/DMEM at the lower chamber for 72h (manufacturer's instrutions). Do you think that this might work? It is worth seeding 150,000 cells which will probably cover the surface completely and possibly lead to clump formation? I believe that cells should be 1-2 cells apart, am I right? 

I know that my cells have a low invasive potential but there are many relevant published articles that have performed invasion assays according to my initial experiments! That is why I don't get it!  Could it be that my cell lines do not invade this particular matrix? Few articles have used the same kit but their protocol in not in detail.

 I am out of ideas and filled with questions, so I would sincerely appreciate your feedback on the matter. 

are you using a transwell system? how do you coat your wells?

Hello Inmost sun,

Thank you for your answer. Yes I am using a transwell system. In fact, I am using an invasion assay kit. The wells are coated with ECMatrix (reconstituted basement matrix of proteins derived from the Engelbreth Holm-Swarm mouse tumor). 

my experience is that the coat is an important thing in that kind of assay; maybe the coat of the kit doesn´t match the needs of your cells; see what ECM has to do with migration of cells ;)

take uncoated transwells and coat yourself, testing various coats

Thank u for your input Inmost sun. This is one of my concerns too. The question is how come this kit has worked in other people's hands, using the same cell lines... It is possible that cell properties differ that much within labs? I 'll have to check that.

if you have really the exact conditions as others who work successfully with that kit it would be a mystery why it does not work in your hands; but see, reproduction of experiments with exact conditions especially compared to other labs are difficult to get in life sciences...