Chemotaxis Assay for Primary Mouse Peritoneal Cells - (Aug/30/2006 )
I am having trouble getting freshly isolated peritoneal cells to migration to chemokines in 96wp chemotaxis chamber (Chemotx, 5um pore). I have no problem doing this sort of assay with cultured cells (i.e. Jurkat, THP-1). I have tried different media: RPMI, DMEM, XVIVO10. But very few cells make it to the lower chamber, and just barely above the back ground (maybe 1% migration even with 100nM concentrations of SDF1a, RANTES or MCP-1). I don't treat the cells or coat the membrane. I usually let the assay go for 1.5h.
Any suggestions would be greatly appreciated.
Are you using the murine chemokines ? I'm guessing you have the human proteins because you've used Jurkats and THP-1s. It might not be the reason because there may be sufficient homology/structural similarity not to effect the binding affinity. To be on the safe side you could try the murine version of one of them. R&D or Peprotech sell them.
All the best,
All the best,
Your point is a good one, but I am using murine cc's. Although for SDF1a and RANTES, the chemotactic cross-reactivity (m and h) is very high.
Just checking they were murine chemokines.
Maybe the resident peritoneal cells won't migrate without activation. Do they express high levels of chemokine receptors? Have you tried cells from whole blood or thioglycolate elicited peritoneal macrophages? My boss have worked on chemokines for a while so I'll ask him next week when he's back at work.
I wonder if using CCR7 ligands might be worth a try. In a model of in vivo peritoneal inflammation the resident peritoneal cells (mostly macrophages) are "lost" initially so whether they migrate to the lymphoid nodes/circulation or apoptose we don't know. Rantes and MCP-1 are produced intraperitoneally but maybe a feature of the resident peritoneal cells is not to migrate to inflammatory chemokines?
Thanks Ceri...Your point about the activation status is a good one. I'll try using elicited PEC's next time.
one of my colligue uses paretoneal macrophages for migration assay. he stimulate with thioglycolate then isolate macrophages so that he can have suffice number of cells for his experiments.
we're having similar problems, did using elicited macrophages help?