Hello,
I am working on a Transwell assay, and I have been trying to optimize the assay for a long time, currently, I am working on hCMEC/D3, seeded at 2x10^4 and left for 5 days (the experiment day), I only change the media once at day 4, but I see this clump only on the lower side of the transwell, could someone help me to figure out why this is happening? ( I am not doing co-culture)
Please see the picture
I have no experince with this specific cell line, however if the cells are culture in the upper compartment and the clump is below the membrane my first guess would be that it is some sort of secreted cellular product (e.g. mucin). Otherwise you can try to reduce the cell number and check if the "clump" appears before the cells reach confluence. If it is only present after the cells have grown to a confluent monolayer it could be cells that are migrating through the porous membrane (if they are small enough).
If you are working with bacterial in the inserts, i suggest that the clumps are bacterial colonies which got through the pores of the membrane. Otherwise what i mentioned above.
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