Protocol Online logo
Top : Forum Archives: : Molecular Biology

NF-kB binding activity in neutrophils and mononuclear cells - (Nov/07/2008 )

I'm trying to stimulate human mononuclear cells and neutrophils with LPS to study the NF-kB binding activity by EMSA but I haven’t been successful yet. Can someone give me a protocol of this method? In my assay I isolate the cells from the blood, then I ressuspend them in Hank’s Balanced Saline Solution and let them stabilize for 30 min in a water bath at 37 ºC with soft agitation, and then I incubate the cells with LPS at different times. But I still can’t see an increase in the signal of EMSA. What can I be doing wrong?

-nocas gomes-

If you are sure about your EMSA procedure and you believe that theproblem is with the stimulation of NFkB, then tray to incubate the cells in a completed cell culture media (e.g. RPMI 1640 supplemented with 10% fetal bovine serum, and antibiotics) in a cell culture incubator (humidified with 5% CO2). Then you can stimulate your cells overnight with LPS.

I hope this will help,

Good luck.