Cell Biology

Stimulation of G protein-coupled receptors (GPCR) by hormones and neurotransmitters elicits cellular responses, many of which result from alterations in the concentrations of cytosolic cAMP and Ca²⁺. Here, we describe a microplate reader fluorescence resonance energy transfer (FRET) assay that uses the genetically encoded biosensors H188 and YC3.60 so that it is possible to monitor the kinetics with which alterations of [cAMP] or [Ca²⁺] occur in monolayers or suspensions of living cells exposed to GPCR agonists. This protocol uses HEK293 cell lines doubly transfected with a FRET biosensor and a recombinant GPCR of interest (e.g., glucagon receptors, CCK₂ receptors, or NPY2R receptors). The protocol allows for rapid screening of small molecule GPCR agonists and antagonists, and it is also useful for discovery of synthetic mono-, dual-, and tri- agonist peptides with GPCR activating properties.

T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand interaction durations impacted on T cell activation. Our newly created opto-ligand-TCR system allowed us to precisely and reversibly control ligand binding to the TCR by light illumination. This system uses phytochrome B (PhyB) tetramers as a light-regulated TCR ligand. PhyB can be photoconverted between a binding (ON) and non-binding (OFF) conformation by 660 nm and 740 nm light illumination, respectively. PhyB ON is able to bind to a synthetic TCR, generated by fusing the PhyB interacting factor (PIF) to the TCRβ chain. Switching PhyB to the OFF conformation disrupts this interaction. Sufficiently long binding of PhyB tetramers to the PIF-TCR led to T cell activation as measured by calcium influx. Here, we describe protocols for how to generate the tetrameric ligand for our opto-ligand-TCR system, how to measure ligand-TCR binding by flow cytometry and how to quantify T cell activation via calcium influx.

Hydrogen sulfide (H₂S) gas is produced in cells and tissues via various enzymatic processes. H₂S is an important signaling molecule in numerous biological processes, and deficiencies in endogenous H₂S production are linked to cardiovascular and other health complications. Quantitation of steady-state H₂S levels is challenging due to volatility of the gas and the need for specialized equipment. However, the capacity of an organ or tissue extract to produce H₂S under optimized reaction conditions can be measured by a number of current assays that vary in sensitivity, specificity and throughput capacity. We developed a rapid, inexpensive, specific and relatively high-throughput method for quantitative detection of H₂S production capacity from biological tissues. H₂S released into the head space above a biological sample reacts with lead acetate to form lead sulfide, which is measured on a continuous basis using a plate reader or as an endpoint assay.

Cyclic adenosine monophosphate (cAMP) is an intracellular signaling messenger derived from the catalytic conversion of ATP, and is a major product of activated G_s protein-coupled receptors. Conversely, formation of cAMP is inhibited by G_i protein-coupled receptors. This protocol has been optimized for the detection of ligand-mediated cAMP accumulation in adherent immortal cell lines expressing G_s-coupled receptors.