I'm doing transmigration assays in which I coat the transwells with HUVEC cells, add different chemokines to the lower chamber and the cells from mouse peritoneum (PEC) on top of HUVEC, and let them migrate through HUVEC toward chemokines. I expected the PEC to migrate more through HUVEC when there is a specific chemokine in the lower chamber compared to the wells where there is no chemokine. I did this experiment three times and for a specific chemokine, SDF-1alpha, less migration could be seen compared to the background.
Does anybody have any idea what would be the reason? I'm somehow sure that there is no problem with HUVEC, because PEC could migrate toward MCP-1 well.
Have you checked whether your HUVECs respond to SDF-1alpha by a change in cell-cell junctions or better yet, a change in vascular permeability? You might assay this in a simpler system without PECs to make sure your can observe activity of your SDF-1alpha preparation. If no change in HUVEC monolayer structure or function can be observed, this may explain the absence of a migration response in the PECs. Some optimization of SDF-1alpha dose may be required, or it may need to be added in conjunction with another vasoactive compound.
In addition, some general notes on transwell assays. First, transwell assays often work best if you coat the bottom side with ECM, so migrating cells stick once they cross the membrane. Second, migration is often assayed in the context of a serum gradient which helps bias the system towards directional migration. Perhaps you are already employing these manipulations.
Thanks Daniel, that seems reasonable, I have to check SDF-1 effect on HUVEC. And for counting migrated PECs, I label them, and then measure the transmigrated cells. And would you please explain more your last sentence? *migration is often assayed...*
Good luck
Sahar- I meant that serum is included at the bottom of the transwell, with your chemokine (such as SDF-1a), and either omitted from the top of the well, or used at a reduced concentration there in the basal medium.
Thanks again. I'll post the checking results here as soon as I get them.
Good luck with your experiments.
as well as the experimental setup suggestions above, there is a process of Fugetaxis where cells can migrate away from chemokines depending on numerous factors such as concentration etc. there is a nice methods paper....http://www.ncbi.nlm.nih.gov/pubmed/20379872
good luck
Ian
Dear Sahar,
May I know the pore size of the transwell inserts you were using?
Mallu, those are 5uM. But, I'm going to use BSA in the lower chamber, because we thought chemokines attach to the plastic of the chamber and therefore will be unable of attracting cells. Do you have any recommendation?
Thanks in advance
Sahar,
thanks for the reply..even I started doing this experiment very recently and I could use some advice myself..I use 8 uM pore filters though.
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