Flow cytometry‐based quantification of cyclic AMP in primary human neutrophils.

Cyclic 3′, 5′adenosine monophosphate (cyclic AMP, cAMP), a ubiquitous second messenger molecule, plays a central role in signal transduction, a large number of cellular functions and drug responses. Although several methods are used to quantify cellular cAMP, such approaches often lack sensitivity and can be difficult to apply to primary cells. Furthermore, currently available assays often require cell lysis and/or large cell numbers to generate readily detectable signals. Here, we describe a fluorescence‐based flow cytometry approach that enables quantification of cAMP even at low cell numbers. In this method, cells are fixed and permeabilized prior to staining using a primary antibody against cAMP and a fluorescent secondary antibody. Use of this method can quantify cAMP levels in freshly isolated human neutrophils, key mediators of the innate immune response and the most abundant phagocytic leukocytes in circulation. The method reproducibly detects and quantifies basal cAMP levels, in addition to cAMP stimulated by forskolin or agonists/antagonist of several Gs/Gi‐linked G protein‐coupled receptors. This approach, in addition to providing a rapid and simple new strategy for the quantification of cAMP, has the potential to facilitate receptor occupancy‐response relationships in terms of second messenger levels and other readouts that can be detected by flow cytometry. Support or Funding Information Supported by NIH grants R56AI110505, T32HL098062 and T32GM007752.