We are trying to establish a system for the thp-1 and u937 transmigration through endothelial cells. However, it seems like none of the cells migrate. We use 8uM pore size. We treat cells with TNF and even use MCP-1 in the lower chamber, but there are no much cells that migrate. We use fluorescent dye to detect the cells. Could it be that cells stick to the bottom of the well or stack in the collagen? Any suggestions?
You might try a smaller pore size, such as 5 or even 3 um. The larger the pore size, the more endothelial cells you need to plate/longer they need to grow to form a confluent monolayer. I seed 40,000 endothelial cells on 3 um pores and let them grow for 4-5 days to become confluent, making sure to change the media on the 2nd or 3rd day. Another issue, make sure your THP-1 cells express CCR2, the receptor for MCP-1. I've found that while some THP-1 lines express CCR2, others don't. You also might try a more generalizable agent, such as FMetLeuPhe, as a chemoattractant. Finally, try varying the number of THP-1 you add for the transmigration and perform a kinetics analysis to determine the optimal time of the assay. Good luck, hope this helps!
Hi Gila, I have the same problem; it may be possible that these cells need to be differentiated to engage in migration. Vitamin D3 or PMA are commonly used for THP-1s. As for U937s, I quit working with these shortly after I saw no migration. Again, they may need to be differentiated. Also, somebody mentioned that THP-1 become pretty much useless after 25 passages. Good luck.
To my experience PMA stimulated THP-1 cells transmigrate through 3 micron trans-membrane coated with human primary aortic endothelial cells from Lonza. The migration pattern varies if the EC monolayer is pre-treated with LPS or oxLDL.
Concern is that physical barrier of a 3 micron membrane might influence the migration significantly. Because of the logically 8 micron would be a better choice, provided you have a tight endothelial monolayer.
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