Enzyme immunoassay kit

Culture supernatants from skimmianine (10, 20 and 30 μM)-treated and LPS (100 ng/mL)-stimulated BV-2 microglia were analysed for levels of PGE₂ using an enzyme immunoassay kit (Arbor Assays). Briefly, 100 µL of samples and standards were added to wells pre-coated with goat anti-mouse IgG. Subsequently, 25 µL of PGE₂ conjugate and PGE₂ antibody were added and incubated at room temperature for 2 h with shaking at 250 rpm. Thereafter, the wells were washed to remove unbound proteins. This was followed by the addition of 100 µL 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) substrate to each well and incubation for 30 min at room temperature. The absorbance was measured in a microplate reader (Infinite F50, Tecan, Männedorf, Switzerland) at a wavelength of 450 nm following the addition of a stop solution.

Urinary ANP was measured using an Enzyme Immunoassay Kit (catalog no.: K026-H1; Arbor Assays, Ann Arbor, MI), and creatinine was measured using the DetectX Urinary Creatinine Detection Kit (catalog no.: K002-H5; Arbor Assays) for pre-injury and 6-wpi time points.⁹ Urine samples were diluted at 1:5 for ANP and 1:20 for creatinine and then plated in a 96-well plate in duplicate. ANP and creatinine plates were read at a 450-nm optical density using SoftMax Pro software (Molecular Devices, LLC. San Jose, CA). Urinary creatinine levels were used to control for differing urine concentrations per ANP enzyme-linked immunosorbent assay (ELISA) kit instructions. To obtain accurate urinary ANP levels, ANP levels were divided by creatinine levels.
Baseline and terminal levels of serum AVP were determined using an arginine vasopressin ELISA kit (catalog no.: OKEH02585; Aviva Systems Biology, San Diego, CA). Stored serum samples (see above) were diluted at 1:5, and ELISA was carried out according to kit instructions.

Rats were killed by decapitation as described above. Trunk blood samples were collected in heparinized test tubes and immediately centrifuged at 850 g for 10 min at 4°C. Plasma samples were then stored at -80°C until analysis. Plasma corticosterone (pCort) was measured in duplicate in 20 μl of plasma by an enzyme immunoassay kit (Arbor Assays, Ann Arbor, MI, United States), as described in detail previously (Puzserova et al., 2013b (link)).

Plasma CORT levels were determined using a commercially available enzyme immunoassay kit (Arbor Assays, Ann Arbor, MI, USA, Catalogue Number K014-H5; sensitivity 20.9 pg/ml) according to the manufacturer’s instructions.