Biotek synergy h1 hybrid plate reader

Cell lines and cell culture: cAMP Hunter^TM Chinese hamster ovary cells (CHO-K1) that express human μ-opioid receptor (OPRM1), human κ-opioid receptor (OPRMK1), and human δ-receptor (OPRMD1) were purchased from Eurofins DiscoverX (Fremont, CA, USA). Cell culture was performed as previously described [18 (link)]. Briefly, cells were plated in a 384-well white tissue culture microplate at 10,000 cells/well density and incubated overnight at 37 °C in 5% CO₂. Compounds were first dissolved in DMSO to form stock solutions (5 mM concentration), and then 9 doses of 100× solutions were prepared by serial dilution with DMSO. Then, 5× solutions were prepared by diluting 100× solutions with buffer consisting of Hank’s Buffered Salt Solution, HEPES, and forskolin. In the agonist assay, cells were treated with compounds (at 1× final concentration) and incubated at 37 °C for 30 min. In the antagonist assay [17 (link)], cells were pretreated with compounds for 15 min at 37 °C followed by 30 min incubation at 37 °C with selected agonists at their EC₅₀ or EC₉₀ dose. The HitHunter cAMP Assay for Small Molecules by Eurofins DiscoverX (Fremont, CA, USA) was then used according to manufacturer’s directions and the BioTek Synergy H1 hybrid plate reader (BioTek, Winooski, VT, USA) and Gen5 Software version 2.01 (BioTek, Winooski, VT, USA) were used to quantify luminescence [19 (link)].

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated ROS production was measured either by a 2′,7′-dichloro dihydrofluorescein diacetate (DCFH-DA) plate assay (28 (link)) or a luminol-based chemiluminescence microtiter plate assay (29 (link), 30 (link)). Also, 2.5 × 10⁴ neutrophils per well were incubated without or with stimulants mentioned above in dark 96-well microtiter plates with 25 μM DCFH-DA (Sigma-Aldrich), which reacts with ROS species produced in intracellular compartments (granules or phagosomes). Fluorescence was recorded immediately in a Biotek Synergy H1 Hybrid plate Reader (Biotek) for 30 min. The response was expressed as relative fluorescence units (RFU). Similarly, 2.5 × 10⁴ neutrophils per well were incubated without or with the aforementioned stimulants in white 96-well microtiter plates with 60μM luminol (5-amino-2,3-dihydro 1,4-phthalazinedione). Chemiluminescence was recorded every 5 min over a period of 30 min in a Biotek Synergy H1 Hybrid plate Reader (Biotek), and the response was expressed as relative luminescence units (RLU).

Cell lines and cell culture: cAMP Hunter^TM Chinese hamster ovary cells (CHO-K1) that express human μ-opioid receptor (OPRM1), human κ-opioid receptor (OPRMK1), and human δ-receptor (OPRMD1) were purchased from Eurofins DiscoverX (Fremont, CA) and used for the forskolin-induced cAMP accumulation assays [28 (link)].
Briefly, cells were plated at 10,000 cells/well density in a 384-well tissue culture plate and incubated overnight at 37 °C in 5% CO₂. Stock solutions of compound were made in 100% DMSO at a 5 mM concentration. A serial dilution of 10 concentrations was made using 100% DMSO, creating 100× solutions of the compound for treatment. The 100× solutions were then diluted to 5× solutions using assay buffer consisting of Hank’s Buffered Salt Solution, HEPES, and forskolin. For the agonist assay, cells were incubated at 37 °C with compounds for 30 min at a 1× concentration. For the antagonist assay, cells were incubated at 37 °C with compounds for 15 min before 30-min incubation at 37 °C with selected agonist at their EC₅₀ or EC₉₀ dose. The HitHunter cAMP Assay for Small Molecules by DiscoverX was then used according to manufacturer’s directions and the BioTek Synergy H1 hybrid plate reader (BioTek, Winooski, VT, USA) and Gen5 Software version 2.01 (were used to quantify luminescence (BioTek, Winooski, VT, USA) [17 (link)].