I am trying Matrigel invasion assay for myeloma cell lines. I am using 8 microns membrane and coating the matrigel at 1: 3 conc. But am not getting results. I am using U266 cell line which is reported to have invasive potential when checked with IGF-1. But I am not getting any results in 48 hours. Some of the papers show pore size 10-12 um. I wonder if this could be a reason?
HI, what is your media composition and what is your concentration of matrigel (5mg/ml)? - we dilute it to 1 mg / ml. Also, we use a high FCS (up to 20%) as the chemoattractant in the lower chamber. I think 8 microns should be fine, it's what we have used in the past. What controls have you got, beads or another invasive line?
I would probably first test your chemoattractant concentrations in a chemotactic migration assay without matrix. I would also lower the Matrigel to a dilution of 1:20 or 1:50, coat for 1hr at 37C and then run the invasion for 24 or 48 hrs.
Thanks all for your suggestions. In fact I am using IGF(5ug/ml) and 20% FCS containing RPMI as chemoattractants. the composition of my matrigel is 1:3 as lower then that makes it very liquid kind of jelly. I am using U266 as a positive control and cells without chemoattractants in the lower chamber serve as treatment control. I will try lower conc of matrigel and will update if it works.
Thanks
Hi Sheerien Rajput , I know this thread was 4 years ago, but did you manage to get ur invasion assay with U266 cells to work? I'm currently trying to set up migration and invasion assays with U266 cells and any advice would be appreciated. Thanks!
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