Detection of Mu‐Opioid Receptors in SH‐SY5Y Human Neuroblastoma Cells by Flow Cytometry

Abstract

PurposePharmacological actions of morphine are mediated through G‐protein‐coupled receptors (GPCRs), specifically mu‐opioid receptors (MORs). Morphine‐induced activation of GPCRs inhibits adenylyl cyclase and decreases 3’,5’‐cyclic adenosine monophosphate (cAMP). Neuroblastoma SH‐SY5Y cell lines endogenously express MORs. Our aim was to develop a flow cytometry (FC) assay for MORs on the surface of SH‐SY5Y cells. This assay will provide in‐depth understanding of opioid‐induced downstream cAMP signaling.MethodsSH‐SY5Y human neuroblastoma cells (ATCC® CRL2266™) were grown in DMEM/10% FBS and maintained in a humidified incubator at 37ºC/5%CO2. Cells were seeded in 96‐well white, opaque‐bottom plates (5×104cells/well). Transfection conditions were optimized using ViaFect™ (Promega™) and a stably transfected SH‐SY5Y cell line was established using GloSensor™ ‐23F cAMP plasmid (Promega™) containing hygromycin‐resistance gene. Cells were washed in FACS buffer (PBS + 2% FBS) and incubated for 1h at room temperature with monoclonal rabbit anti‐MOR antibody (Ab) (Novus Biologicals). Following the wash, cells were incubated with donkey anti‐rabbit secondary Ab (Abcam). We used monoclonal mouse anti‐muscarinic Ab (Novus Biologicals) and donkey anti‐mouse secondary Ab (Jackson Labs). Both secondary Abs are bound to a fluorophore (Alexa Fluor® 488) which emits light when excited by FITC channel frequency (excitation/emission: 495nm/519nm). A Cytoflex flow cytometer (Beckman Coulter) analyzes cells for FITC fluorescence emission. Multiple primary and secondary concentrations were tested; Human TruStain Fc blocker (BioLegend) was used to prevent nonspecific tagging.ResultsRelative fluorescence emission was measured as percent shift increase from the untagged control group. Because MOR Abs have been tested in a limited capacity, we tested for presence of muscarinic receptors (another endogenously expressed GPCR in these cells) to establish the FC assay. Initial trials with low primary and secondary MOR Ab concentrations showed limited shifts (~1‐2%), however, muscarinic receptor Abs bound to their receptors at lower concentrations compared to MOR Abs. Testing muscarinic receptors showed secondary Ab concentration‐dependent fluorescence shifts indicating that the technique was viable. Studies with increased concentration ratios of primary and secondary MOR Abs showed secondary Ab concentration‐dependent response with maximum % shifts reaching up to 16%. Use of Fc blocker eliminated nonspecific binding, increasing the percent shift.ConclusionCompared to untagged controls, antibody‐tagging on SH‐SY5Y cell surface produced greater immunofluorescence indicating presence of MORs in both non‐transfected and 23F cAMP plasmid transfected cells. Direct binding of receptor modulators leads to altered downstream cAMP signaling as shown in previous in vitro signaling studies and behavioral mouse models. Detection of the MOR is essential to ensure that cAMP signaling is regulated by MOR agonists. In future studies, we plan to investigate the expression of endothelin‐A receptor (ETAR) and explore potential interactions between MOR and ETAR in SH‐SY5Y cells.