Scintillation spectrometry

We have reported on the effect of hypoxia on PAFR binding to FPASMC, but have studied newborn PASMC during normoxia only. Therefore, we investigate, in parallel, the effect of normoxia, hypoxia, and hyperoxia on PAFR binding to newborn PASMC. Receptor binding assays were performed in conditions of normoxia and hypoxia as previously reported by us (Ibe et al. 2005; Renteria et al. 2010, 2013b), and a third treatment in hyperoxia condition as described above. Briefly, the cells were washed with calcium‐ and magnesium‐free PBS before use according to the specific study protocol. After incubation in normoxia, hypoxia, or hyperoxia, unbound ³H‐PAF was washed off with ice‐cold PBS, and then incubated on ice for 30–45 min in saline/EDTA mixture containing 154 mmol/L saline and 5 mmol/L EDTA. Receptor‐bound ³H‐PAF was extracted on Whatman GF/C membrane filters using in‐line vacuum system and then subjected to extraction procedures as previously reported (Ibe et al. 2000, 2002, 2005, 2007). Cell‐bound PAF radioactivity was quantified by scintillation spectrometry (Beckman Instruments, Fullerton, CA). In studies probing the interaction of PAF with its receptors in the presence of other agonist or antagonists, cells were preincubated with the agent before the addition of ³H‐PAF, and then incubated further according to the specific experimental protocol.

5-HT_2A binding was analyzed in membrane preparations as previously described (Canal et al., 2010 (link)). Three or 10 days following mTBI or sham treatments, subjects were sacrificed via rapid decapitation and bilateral frontal cortex samples were dissected on ice. Samples were homogenized in 3 ml ice-cold Tris binding assay buffer (50 mM Tris-HCl, 10 mM MgCl₂, 0.1 mM EDTA, pH = 7.4), and subsequently centrifuged at 20,000 x g for 20 min at 4°C. The supernatant was decanted, and the pellet was resuspended in a 1.5-ml binding assay buffer. Protein concentrations were measured with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Membrane preparations (200 µg protein) were incubated with the 5-HT_2A antagonist [³H]ketanserin (PerkinElmer, Waltham, MA) (1 or 10 nM) at 37°C for 60 min. Samples were run in duplicate and nonspecific binding was determined in the presence of 100 µM methysergide (Sigma Aldrich, St Louis, MO). Samples were collected using a Brandel cell harvester and washed with ice-cold phosphate-buffered saline (PBS, pH = 7.4). Samples were incubated in 7 ml scintillation fluid overnight (National Diagnostics) and radioactive counts were measured via scintillation spectrometry (Beckman Coulter).