Hi all, using a transwell assay to test migratory function of cytokines, Costar transwells 5 micrometer, 24 well, according to publications and protocols 100 ul in top compartment with 1x10*6 cells (huPBMCs)/ml and bottom with 600 ul media10%FCS +/- cytokine.
Time titration 2-4 hr, and all I see in cytokine as well as in the media only control is cell debris.
Any advice? Very appreciated. Cheers, Axel
Sorry to hijack you rpost - but it's kinda related - I get cell migration through of human PBMC< but I get the same numebr in the control (RANTES) and the negative control media only with FCS. I have been told to reduce the FBS, or subsitute BSA instead, but I can't do that or it disturbs another part of the experiment.However it can mask the effects of some chemokines. I would try it without the FCS is you can.
How are you counting the cells that migrate? Manually or with a cell counter?
Have just come across this The most frequent causes of zero migration are:
The cells are either dead or inactive. Contamination of the gasket is one possible cause of cell death. Replacement Gaskets are available for our chambers. Many researchers keep a spare gasket on hand.
Either the chamber is not assembled properly, or the lower wells are not filled sufficiently, so the fluid in the lower wells does not contact the bottom surface of the filter.
The pore size is too small to allow migration of the cells. We offer filters in a wide range of pore sizes to optimize your assay.
The blue separator paper has been placed in the chamber rather than the (translucent) polycarbonate filter.
When you have active cells and an appropriate filter contacting the fluid in the lower wells, there should be at least some random migration. Inactive chemokines may result in little or no differential between test sites and negative controls, but will not normally result in zero migration.
I am using 8um pore size, but I've read papaers that used 5um, so I don't think is the problem
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