Blood samples were collected in ice-chilled tubes containing ethylenediaminetetraacetic acid and aprotinin. Plasma obtained by centrifugation (3000 g) was stored at −80°C until assayed for HPA output, including adrenocorticotropin (ACTH) and corticosterone, in addition to sex steroid hormones. Estrous cycle phase in females was not determined in the current study; however, gonadal status (testosterone in males and estradiol in females) was assessed from trunk blood samples obtained during tissue harvesting. Plasma ACTH, testosterone, and estradiol were measured using commercial radioimmunoassay (RIA) kits (MP Biomedicals, Solon, OH, USA). Corticosterone was measured by RIA as previously described (Viau and Meaney, 1991 (link)). Briefly, equal volumes of plasma sample, [3H]corticosterone (3 nmol/l PerkinElmer, Waltham, MA, USA), and primary antiserum (1:2500, Ab1297, Sigma, Boston, MA, USA) were incubated overnight at 4°C. Dextran-coated charcoal was then added to the incubates at 4°C, after which bound ligand was obtained by centrifugation (3000 g) for 10 minutes. Intra- and inter-assay coefficients of variation were 6% and 12%, respectively, for ACTH; 8% and 10% for corticosterone; 3% and 8% for testosterone; and 6% and 11% for estradiol. Detection limits were 10.0 pg/mL (ACTH), 10 ng/mL (corticosterone), 0.1 ng/mL (testosterone), and 1.5 pg/mL (estradiol).
3h corticosterone
Manufactured by PerkinElmer
Sourced in United States
[3H]corticosterone is a radiolabeled form of the steroid hormone corticosterone, which is used in research applications. It contains the radioactive isotope tritium (3H) incorporated into the chemical structure of corticosterone. This product can be used in various biochemical and physiological studies that require the detection or quantification of corticosterone.
Automatically generated - may contain errors
Lab products found in correlation
3h cortisol, by PerkinElmer (2 mentions)
Ria kit, by MP Biomedicals (1 mentions)
Corticosterone, by Beckman Coulter (1 mentions)
Ls 6500 multi purpose scintillation counter, by Beckman Coulter (1 mentions)
Corticosterone, by Merck Group (1 mentions)
Ecoscint h, by National Diagnostics (1 mentions)
Tri carb 2900tr liquid scintillation analyzer, by PerkinElmer (1 mentions)
10 protocols using 3h corticosterone
1
Plasma Hormone Measurements in Rodents
2
Circadian Plasma Corticosterone Measurement
3
Restraint Stress and Plasma Corticosterone
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Blood was collected via the tail vain at the start of restraint stress (placement in a 50 ml plastic conical tube) and after 30 minutes of restraint stress. Plasma corticosterone concentrations were measured by RIA. Plasma samples were diluted 1∶25 in 0.01 M PBS, and corticosterone binding globulin was denatured by incubating the samples at 65°C for one hour. All samples and standards were then incubated overnight at 4°C in the presence of antiserum (MP Biomedicals, Solon OH) and [3H] corticosterone (Perkin- Elmer, Boston, MA) in 0.1% gelatin dissolved in 0.01 M PBS. Unbound corticosterone was removed by adding dextran-coated charcoal, which was then separated by centrifugation. Bound corticosterone was decanted into new vials, mixed with scintillation fluid, and counted using a Beckman Coulter LS6500 Multipurpose Scintillation Counter (Brea, CA). Plasma corticosterone concentrations were determined by comparison to a standard curve (5–700 ng/ml). The intra-assay variance was 3.26%, and was determined by an internal control that was measured at regular intervals throughout the assay.
4
Corticosterone Quantification in Rat Serum
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For Corticosterone quantification, rats were anesthetized with isoflurane mixed
with medical air and 0.5 mL of whole blood was quickly collected from the tail
vein on day −1 and 6. Samples were always collected at 10 a.m. The whole blood
was allowed to cloth for 15 min and centrifuged at 6000 r/min (3.5 g) for 8 min
at room temperature to obtain serum samples. Samples were stored at −20℃ until
further analysis by radioimmunoassay. Corticosterone (Sigma Chemical Co., MO,
USA.) was used as standard and 3H-Corticosterone as tracer (Perkin
& Elmer, MA, USA). The sensitivity of the assay was 3 nM. The intra- and
inter-assay variations were 6% and 9.6%, respectively.
with medical air and 0.5 mL of whole blood was quickly collected from the tail
vein on day −1 and 6. Samples were always collected at 10 a.m. The whole blood
was allowed to cloth for 15 min and centrifuged at 6000 r/min (3.5 g) for 8 min
at room temperature to obtain serum samples. Samples were stored at −20℃ until
further analysis by radioimmunoassay. Corticosterone (Sigma Chemical Co., MO,
USA.) was used as standard and 3H-Corticosterone as tracer (Perkin
& Elmer, MA, USA). The sensitivity of the assay was 3 nM. The intra- and
inter-assay variations were 6% and 9.6%, respectively.
5
Tissue Explant 11β-HSD Activity Assay
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11β-HSD1 oxo-reductase activity (11DHC to corticosterone) was assessed by incubating freshly dissected tissue explants with 100 nm 11DHC diluted in 1 mL media (in glass tubes) with tracer amounts of [3H]-11DHC (synthesized in-house (Bujalska et al. 2002 (link))) at 37°C for 2 h. 11β-HSD2 dehydrogenase activity (corticosterone to 11DHC) was assessed by incubating freshly dissected tissue explants with 100 nm corticosterone diluted in 1 mL media (in glass tubes) with tracer amounts of [3H]-corticosterone (PerkinElmer) at 37°C for 2 h. Following incubation, steroids were extracted from the medium with dichloromethane, separated by thin-layer chromatography with chloroform/ethanol (92:8), and the fractional conversion of the steroids was calculated by scanning analysis with a Bioscan 2000 radioimaging detector (Bioscan, Washington, DC, USA). Percentage conversion was normalized to tissue weight.
6
Radiolabeled-Steroid Saturation Assay for CBG Quantification
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A radiolabeled-steroid saturation assay was used to detect and measure CBG in
concentrated and buffer-exchanged culture media or after chromatographic purification
(Simard et al. 2015 (link)). In
brief, steroid-binding capacity measurements and Scatchard analyses of steroid-binding
affinity were performed using [3H]cortisol or [3H]corticosterone
(PerkinElmer Health Sciences) as the labeled ligands for human and rat CBGs, respectively,
and dextran-coated charcoal to separate bound from free [3H]-labeled steroids
(Hammond & Lahteenmaki 1983 (link)).
concentrated and buffer-exchanged culture media or after chromatographic purification
(Simard et al. 2015 (link)). In
brief, steroid-binding capacity measurements and Scatchard analyses of steroid-binding
affinity were performed using [3H]cortisol or [3H]corticosterone
(PerkinElmer Health Sciences) as the labeled ligands for human and rat CBGs, respectively,
and dextran-coated charcoal to separate bound from free [3H]-labeled steroids
(Hammond & Lahteenmaki 1983 (link)).
7
Quantifying 11β-HSD Dehydrogenase Activity
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Small pieces of fresh epididymal adipose tissue were manually homogenized on ice, and homogenates were diluted to obtain a final concentration of 0.05 mg protein/mL. The reaction tubes containing 15 nM of mixed cold [3H]-corticosterone (Perkin Elmer) were covered and incubated at 37°C for 10 or 30 min. Steroids were extracted with ethyl acetate, and the dried pellet was resuspended in ethanol. Hormones were separated by thin layer chromatography in silica gel Polygram SIL N-HR/UV 254 (Macherey-Nagel, Düren, Germany) using diclormethane:acetone (4:1) as a mobile phase.
Cold standards of 11-dehydrocorticosterone and corticosterone were used to identify their specific zone migration. Each hormone zone was mixed with scintillation solution Ecoscint H (National Diagnostics, Nottingham, UK) in order to quantify the radioactivity. 11β-HSD dehydrogenase activity was expressed as pmol of 11β-dehydrocorticoterone produced per mg of protein and hour of incubation.
Cold standards of 11-dehydrocorticosterone and corticosterone were used to identify their specific zone migration. Each hormone zone was mixed with scintillation solution Ecoscint H (National Diagnostics, Nottingham, UK) in order to quantify the radioactivity. 11β-HSD dehydrogenase activity was expressed as pmol of 11β-dehydrocorticoterone produced per mg of protein and hour of incubation.
8
Quantifying Human CBG Steroid Binding
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Measurements of the cortisol-binding capacity of human CBG and its steroid-binding affinity by Scatchard analysis were performed using [3H]-cortisol or [3H]-corticosterone (PerkinElmer Life Sciences), as described (Hammond & Lahteenmaki 1983 (link)).
The 12G2 and 9G12 ELISAs of CBG in human blood samples have been described (Lewis et al. 2003 (link), Lewis & Elder 2011 (link)). Blood samples or purified CBG were diluted appropriately, and plasma with a known CBG concentration, based on cortisol-binding capacity, was serially diluted for ELISA standards.
Included within these assays are two reference plasma samples, in which the CBG-binding capacity has been determined. One of the samples is consistently discordant in the 9G12 ELISA, whereas the other is consistently concordant between all assays.
The 12G2 and 9G12 ELISAs of CBG in human blood samples have been described (Lewis et al. 2003 (link), Lewis & Elder 2011 (link)). Blood samples or purified CBG were diluted appropriately, and plasma with a known CBG concentration, based on cortisol-binding capacity, was serially diluted for ELISA standards.
Included within these assays are two reference plasma samples, in which the CBG-binding capacity has been determined. One of the samples is consistently discordant in the 9G12 ELISA, whereas the other is consistently concordant between all assays.
9
Radioimmunoassay for Corticosterone Quantification
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CORT was measured using RIA as previously described27 (limit of detection = 4.45 ng/ml). Plasma samples were diluted 1:25 in 0.01 M phosphate buffered saline (PBS) (pH = 7.4). CORT binding globulin was denatured by heating samples at 65C for 1 h. A standard curve ranging from 2.5 pg to 750 pg CORT (Steraloids) was run alongside all assay tubes. All samples and standards were incubated with CORT antiserum (1:1200, Cat# 7120016, RRID:AB_2801269; MP Biomedicals) and 3H corticosterone (Perkin Elmer) overnight at 4°C. Unbound CORT was removed by adding dextran‐coated charcoal and centrifuging for 15 min at 3000 rpm and 4°C. The supernatant containing bound CORT was decanted into plastic scintillation vials, mixed with 4 ml liquid scintillation cocktail (Ecoscint Ultra; National Diagnostics), and counted using a Tri‐Carb 2900TR (Packard Tri‐Carb 2900TR Liquid Scintillation Analyzer, RRID:SCR_018610; PerkinElmer). Sample CORT concentrations were determined via comparison to a standard curve (2.5‐750 pg) and data was analyzed using Graphpad Prism Software (GraphPad Prism, RRID:SCR_002798). The intra‐assay variance was 9.4% and was determined using internal quality controls measured throughout the assay. All samples from a single experiment were run in a single assay.
10
Hippocampal 11β-Hydroxysteroid Dehydrogenase Activity
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To evaluate 11BHSD activity, an assay mixture was used containing 100 nM 3H-corticosterone (specific activity 16.6 GBq/mmol; Perkin Elmer, MA, USA) in Krebs Ringer buffer (pH 7.4), 2 mM nicotinamide adenine dinucleotide phosphate (NADP), 0.2% glucose, and hippocampal homogenates obtained for western blotting that were diluted with Krebs buffer to 1.5 mg protein/mL. Blanks were included by adding buffer instead of homogenates. The reaction mixes were incubated for 2 h at 37°C. Steroids were extracted using 2 mL ethyl acetate and separated by thin layer chromatography (TLC) using dichloromethane:acetone (4:1) as the mobile phase. The radioactivity in each TLC fraction was measured using standard liquid scintillation. The assay was performed in duplicate for each sample. The activity was expressed as pmol of 11-dehydrocorticosterone per mg of protein and hour of incubation.
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