6500 ls counter

For the SAM uptake assays, tissue homogenates from 129Sv mice treated with tritiated SAM were processed as previously described [17 (link)] to obtain whole tissue lysates. The samples were then mixed with scintillation solution and counted using a Beckmann 6500 LS counter; the results were normalized as cpm/μg of tissue.

Corticosterone was measured by radioimmunoassay following Wingfield et al. 1992^{46 (link)}. An exact volume of plasma was equilibrated with 2000 cpm of tritiated corticosterone (purchased from Perkin Elmer, NET399250UC). Steroids were extracted in redistilled dichloromethane, dried under nitrogen in a water bath at 35 °C, and re-suspended in phosphate buffered saline with 1% gelatin. A 100 µl aliquot was added to a scintillation vial and combined with scintillation fluid (Perkin Elmer Ultima Gold: 6013329) to determine percentage recovered from dichloromethane extraction. Duplicate 200 µl aliquots were assayed by adding 100 µl (~ 10⁴ CPM) of tritiated corticosterone (Perkin Elmer NET399250UC) and 100 µl of antibody (MP Biomedical 07–120016, lot 3R3-PB-20E antibody). Unbound steroids were separated from bound steroids with 500 µl of dextran-coated charcoal, followed by centrifugation at 3000 g for 10 min. The supernatant was decanted and combined with scintillation fluid and counted for 5 min or within 2% accuracy on a Beckman 6500 LS counter. Values were corrected for volume and individual recoveries. Inter- and intra-assay variations were 4.45% and 5.50%, respectively, and mean recoveries were 85.1%.

Plasma CORT was quantified by radioimmunoassay as described and validated for this species (Names et al., 2021a (link)). Reconstituted CORT was assayed in duplicate with tritiated CORT (Perkin Elmer NET399250UC, Waltham, MA, USA) and CORT antibody (MP Biomedicals 07120016, lot 3R3-PB, Solon, OH, USA) and combined with Ultima Gold scintillation fluid (Perkin Elmer 6013329) to be counted for 5 min or within 2% accuracy on a Beckman Coulter 6500 LS counter (Brea, CA, USA). Results were averaged across duplicates and corrected for individual sample recoveries. Mean (±s.d.) recovery was 92.85±2.94%, intra-assay variation (calculated using coefficient of variability, CV, between duplicates) and inter-assay variation (calculated using CV among assay standards) were 5.16% and 5.55%, respectively, the limit of detection was 9.36±0.26 pg (mean±s.d.) per tube, and the mean bound to free ratio was 0.35.