Hi Massimo,

Are you looking for cell surface markers? or intracellular? or you have luxury to do ELISA? For the best marker(s) you need to provide more details.

As a wild suggestion, you can use multiparametric flow cytometry to measure TNFa, IL-1b, IL-6 and Il-8 (intracellular staining in combination with CD14 and CD16, after incubation for 5 hr in the presence of brefeldin A). In parallel, you can also use DCFDA to measure reactive oxygen species.

Hi Sergey,

thanks for your suggestion. I would prefer surface markers, but I can look for intracellular. I use multi parametric analysis by flow cytometry. I would evaluate the activation status of circulating monocytes and neutrophils from subjects exposed to different pro-inflammatory/oxidative stimuli.

Massimo, you may already know that widely used core combination of cell surface markers for human monocytes would be CD14/HLA-DR/CD16 (classical, non-classical and intermediate subsets). I also use CD38 (as a second marker for non-classical subset; CD16 can be down-regulated by pro-inflammatory stimuli). This combination of markers will provide fast answer and well accepted by reviewers:) Pro-inflammatory CD14posCD16high/HLA-DRhigh/CD38neg subset is characterized by higher level of TNFa and IL-8 production (after LPS stimulation) when compared to CD14posCD16negHLA-DRlowCD38pos or CD14posCD16lowCD38pos subsets. The only problem is that pro-inflammatory cytokine secretion is highly variable (even in healthy volunteers) and just weakly correlates with percentage of pro-inflammatory monocytes. This is why I suggested intracellular staining for pro-inflammatory cytokines. However, it takes 7-8 hours.

For neutrophils (CD14lowCD16highHLA-DRneg), which are not great in cytokine production compared to monocytes, I am also using DCFDA staining for ROS (30 min, 1 uM).

Hi Massimo,

I would like to recommend evaluating the expression of CD11b on neutrophil and monocyte. CD11b is Mac-1. The expression of Mac-1 is increased on activated neutrophil and monocyte.

Best,

Kunihiko

A very sensitive cell surface marker of monocyte activation is tissue factor - it appears on the surface of the cells very rapidly and robust increases can be detected..

I sensitive cell surface marker for PMN activation is surface MMP-9. MMP-9 is stored in tertiary granules of PMNs and rapidly appears on the cell surface in large quantities following PMN activation (see PMID 12663332 for methods) .

There are great Abs to human tissue factor and MMP-9 and you can quantify using FACS.