Plasma CORT (pg/ml) was quantified using a validated specific CORT ELISA kit (ENZO Life Sciences - ADI-901-097)50 (link). IFN-γ and IL-4 (pg/ml) were quantified using validated specie-specific ELISA kits (Uscn Life Science Inc., IFN-γ: E90049Ga; IL-4: E90077Ga). The reactivity with CORT was 100% with a sensitivity of 27.0 pg/ml detecting concentration ranging from 32 to 20.000 pg/ml. The cross reactivity of related steroids compound was: deoxycorticosterone 28.6%, progesterone 1.7%, testosterone 0.13%, tetrahydrocorticosterone 0.28%, aldosterone 0.18%, cortisol 0.046, pregnenolone and cortisone <0.03%. The reactivity with IL-4 was 100% with a sensitivity of 15.63 pg/ml detecting concentration ranging from 16 to 1.000 pg/ml. The reactivity with IFN-γ was 100% with a sensitivity of 15.63 pg/ml detecting concentration ranging from 16 to 1.000 pg/ml. No significant cross-reactivity or interference between the analysed cytokines and their analogues was observed. Intra- and inter-assay variability for each target molecule were 8.4% and 8.2% (corticosterone), 9% and 11% (IL-4 and IFN-γ). Determinations were done following the procedure specified by the manufacturer. All samples for each target molecule were assessed the same day and by duplicate.
Cort elisa kit
Manufactured by Enzo Life Sciences
Sourced in United States
The CORT ELISA kit is a laboratory assay designed to quantify the concentration of cortisol, a steroid hormone, in biological samples. The kit utilizes an enzyme-linked immunosorbent assay (ELISA) format to detect and measure the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ, by USCN (1 mentions)
Heparin coated capillary tubes, by Drummond (1 mentions)
Microtainer serum separator tube, by BD (1 mentions)
Rolipram, by Merck Group (1 mentions)
Anti pcreb, by Merck Group (1 mentions)
Anti creb, by Merck Group (1 mentions)
Kt5823, by Cayman Chemical (1 mentions)
Anti rabbit lgg, by MultiSciences Biotech (1 mentions)
11 protocols using cort elisa kit
1
Quantification of Plasma Hormones and Cytokines
2
Stress-Induced Corticosterone Measurement
3
Diurnal Variation in Serum Corticosterone
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All the animals were sacrificed between 10:30 h and 13:00 h, with exception of the PS 6 h group, which was sacrificed between 17 h and 18 h. Trunk blood samples were collected for the determination of serum CORT levels. Hormone level determination was carried out using CORT ELISA Kit (Enzo, New York, USA; Cat. ADI-900-097), according to the kit’s instructions. CORT levels were not evaluated in animals that were assessed for the OLT.
4
Restraint Stress Induces Corticosterone
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During the animal’s light cycle between 10:00 and 13:00, single-housed mice were restrained for 15 min in modified 50 ml conical tubes with the cone endings removed and an aperture added to the cap for the tail. Tail blood was collected with heparin-coated capillary tubes (Drummond) at time points 0, 15, 30 and 90 min from the onset of restraint stress. Plasma was collected from blood by centrifugation at 2,000× g for 10 min. CORT levels were measured in plasma using CORT ELISA kit (Enzo Life Sciences). Using 5 μL of plasma, samples were prepared and analyzed in duplicate on a 96-well plate following manufacturer’s protocol.
5
Serum CORT Level Determination
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Trunk blood samples were collected for the determination of serum CORT levels. Special care was taken to avoid pre-decapitation stress while decapitation took place, so the other animals were left outside the room during this process. Blood was then centrifuged at 4000× g for 15 min, and serum was collected and stored at −20°C. Hormone level determination was carried out using CORT ELISA Kit (Enzo, New York, NY, USA; Cat. ADI-900-097), according to the instructions provided by the kit.
6
Serum Cortisol Extraction and Quantification
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For this procedure, blood samples (~0.5 mL) were collected from the facial vein, kept at room temperature (30 min), and then centrifuged (6,000 rpm, 10 min, 4°C), the supernatants were collected and re-centrifuged (6,000 rpm, 10 min, 4°C), and the sera were collected and stored (−80°C). CORT ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA, Cat #ADI-901-097) was used for CORT quantification, following the manufacturer’s specifications. To avoid inter-assay variance, we analyzed all the samples in a single run.
7
Corticosterone Stress Response in Mice
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A total of non-stressed (10 mice per genotype) and stressed (9–10 mice per genotype) mice were used for corticosterone (CORT) measurements. Each group contained a balanced number of males (~5) and females (~5). Blood from wild type and Mapt−/− mice was collected via the submandibular vein one hour following the start of the light cycle (basal) and 30 min following a 10 min tube restraint (stressed) [30 (link)]. Serum was separated from whole blood using microtainer serum separator tubes (BD, Franklin Lakes, NJ, USA) and centrifuged for 15 min at 1300× g. Levels of serum CORT were quantified using a CORT ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA), as recommended by the manufacturer.
8
Chronic Stress Impacts on Offspring Behavior
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Following the final chronic variable stress exposure, mice were pair housed with purchased 8-week-old B6 females for 2 weeks. This 2-week period was chosen to: (a) control for any effects of acute stress prior to breeding on maternal care for the offspring; and (b) allow eventual sires to purge older sperm that matured prior to the onset of chronic stress exposure. Plasma CORT levels were measured 1 week following the final stressor and 2 h before the start of the dark cycle (17:00) using the CORT ELISA kit (Cat# ADI-900-097; Enzo Life Sciences, Farmingdale, NY, USA). After the 2-week post-stress period, all males were moved to housing with two stress-naïve Strain 129 8-week-old females for 48 h before males were removed and pregnant dams were single-housed for rearing of stress (S) and control (C)-sired offspring. Offspring were weaned at 3 weeks postnatal and group-housed (3–4/cage) with same sex littermates of the same treatment group. Importantly, for all behavioral testing, at least one and no more than two mice of the same sex were examined per litter and per sire.
9
Corticosterone Extraction from Egg Yolks
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Corticosterone was assayed to see whether different CP levels would affect stress responses in laying hens. The separated yolks were pooled (3 yolks/replicate) and homogenized, and 4 g of homogenized egg yolks were diluted with an equal volume of phosphate buffered saline in the falcon tube. Then 1 mL of the diluted yolk was mixed with an equal volume of ethanol, and the mixture was extracted at 37°C for 1 h in the incubator and then centrifuged (Kim et al., 2021 ). The supernatants were analyzed using a CORT ELISA kit (Enzo Life Science Inc., ADI-901-097, Farmingdale, NY) per the manufacturer's recommendation.
10
Amyloid-beta Aggregation and Rolipram Treatment
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Aβ1-42 (rPeptide, USA) was dissolved in 0.9 % sterile saline, at a final concentration of 0.4 μg/μl, and incubated at 37°C for 4 days to obtain aggregated Aβ before microinfusion into cerebroventricle (Wang et al., 2012 (link)). Rolipram (Sigma-Aldrich, USA) was prepared by being dissolved in 0.9% sterile saline containing 1% dimethyl sulfoxide (DMSO). One and a half months after microinjection with Aβ1-42, the mice were treated with different doses of Rolipram (0.1, 0.5, 1.0 mg/kg/day, i.p.) or vehicle for 2 weeks. KT5823 (Cayman Chemical, USA), a selective inhibitor of cGMP-dependent protein kinase (PKG), and H89 (Sigm-Aldrich, USA), a cAMP-dependent protein kinase (PKA), were dissolved in artificial cerebrospinal fluid (ACSF) and were bilaterally microinjected into the intracerebroventricular, 30 min before treatment with Rolipram.
The primary antibodies of anti-CRFR1, anti-BDNF, and anti-GR were purchased from Abcam Biotechnology Company (Abcam, Cambridge, MA). The anti-pCREB and anti-CREB were purchased from Merck Milipore (Millipore,Billerica,MA,USA). All the secondary antibodies (anti-rabbit lgG) were purchased from MultiSciences Biotech Co., Ltd. (MultiSciences, Hangzhou, China). The CORT ELISA kit was purchased from Enzo Life Sciences (Enzo Life Sciences, USA).
The primary antibodies of anti-CRFR1, anti-BDNF, and anti-GR were purchased from Abcam Biotechnology Company (Abcam, Cambridge, MA). The anti-pCREB and anti-CREB were purchased from Merck Milipore (Millipore,Billerica,MA,USA). All the secondary antibodies (anti-rabbit lgG) were purchased from MultiSciences Biotech Co., Ltd. (MultiSciences, Hangzhou, China). The CORT ELISA kit was purchased from Enzo Life Sciences (Enzo Life Sciences, USA).
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