Hi,
I am trying to detect cytokines IL-1b, IL-8, TNF-a and IFN-g in porcine monocytes and macrophages by flow cytometry. I have problem with high spontaneous production (without stimulation with addition of brefeldin A) of IL-1b and IL-8 by either monocytes or macrophage. Moreover the production of these cytokines is not stimulable by common stimuli (LPS, IFN-g+LPS, PMA+ionomycin) - there is not difference between unstimulated and stimulated cells. The finding was confirmed by real time PCR. The cytokine staining is specific. I have tried everything - different Lot's of media and FTS, with and without antibiotics, different culture plates (Delta Nunclon or plates with surface which should not bind cells from NUNC), stimulation of isolated mononuclear cells vs. stimulation of the whole blood, gentle manipulation with cells (centrifugation at low speed, gentle mixing). I use sodium heparin. The culture conditions are sterile.
These problems does not occure when IFN-g and TNF-a are detected - spontaneous production is undetectable and stimulated cells produce high amounts of these cytokines.
Moreover this problem does not comprise detection of porcine cytokines only but also detection of iNOS in murine macrophages - very high spontaneous production of NO (Griess reaction), high expression of iNOS (Western Blot, PCR, immunocytochemistry), low difference between unstimulated and stimulated samples.
Nobody other in our lab work with phagocytes, everybody use lymphocytes only and have not problems with spontaneous activation of these cells.
Do have anybody some other idea where can be the problem?
Petra
