Bio plex array reader

Isolated purified EM CD8+ T cells or purified CD103+ and CD103-CD8+ T cells were plated (50,000–200,000 cells/well) in round bottom ultra-low attachment 96-well plates (Corning, Corning, NY) in X-VIVO 15 without Phenol Red media supplemented with 10% charcoal dextran-stripped human AB serum. Purified CD8+ T cells were treated with or without E₂ (5 × 10^− 8 M) or P (1 × 10^− 7 M) for 48 h. Purified CD103+ and CD103-CD8+ T cells were activated with phorbol 12- myristate 13-acetate (PMA) (100 ng/ml, Abcam) and ionomycin (2 μM, Calbiochem) for 24 h. The culture supernatants were collected and stored at − 80 °C until analysis. TNFα, IL-6 and IFNγ were measured using Millipore human cytokine multiplex kits (EMD Millipore. Corporation, Billerica, MA) according to the instructions. Signal was measured using the Bio-Plex array reader (Bio-Rad). Bio-Plex Manager software with five-parametric-curve fitting was used for data analysis. Data were calculated according to the cell number to compare baseline production or normalized as the fold change in baseline production to determine responding to hormone treatment or PMA stimulation.

Plasma choline, betaine, trimethyllysine (TML), butyrobetaine and trimethylamine N-oxide (TMAO), as well as L-carnitine, acetylcarnitine, proponylcarnitine, octanoylcarnitine, iso-/L-valerylcarnitine and palmitoylcarnitine, were analysed in EDTA-plasma using LC/MS/MS as previously described [58 (link), 59 (link)].
Clinical laboratory measurements, including safety parameters were measured in EDTA-plasma using routine methods at the central laboratory at Haukeland University Hospital (Bergen, Norway).
Total AOC of plasma was measured using the total antioxidant capacity kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. The protein mask was not used, enabling the analysis of both small molecule antioxidants and proteins ability to reduce Cu²⁺ to Cu⁺. In brief, EDTA-plasma was allowed to reduce Cu²⁺ for 1.5 hour at room temperature on an orbital shaker. The absorbance was measured at 570 nm using a plate reader. Results were expressed as trolox equivalents according to a trolox standard curve.
The levels of IL-1β, IL-10, IL-17, G-CSF and TNF-α were measured in plasma samples using a custom-made multiplex MILLIPLEX MAP kit (Millipore Corp., St. Charles, IL, USA), and the assay solution was read by the Bio-Plex array reader (Bio-Rad, Hercules, CA, USA) and determined with the Bio-Plex Manager Software 4.1.

Mice were anesthetized and the blood was collected by cardiac puncture at 0.5, 24.5, 48.5, and 72.5 h post‐EP‐HCA1‐PalmGRET injection. The blood of mice with PBS injection were included as the baseline (control). 500–700 µL of mice whole blood was collected in 1.5 mL and allowed to clot at 4 °C for 30 min before centrifugation. After centrifugation with 1500 × g at 4 °C for 10 min, the serum was transferred to a new 1.5 mL microcentrifuge and stored at −80 °C before Multiplex Cytokine Assay. Bio‐Plex Multiplex Immunoassays Assay (Bio‐Rad, California, USA) was performed according to manufacturer's instruction. 50 µL serum was loaded per well into 96‐well microplate, and each sample was test in duplicates. Murine IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐10, IL‐12, IL‐13, IL‐17A, IL‐22, IL‐23, TNF‐α, and IFN‐γ signal were test by the Bio‐Plex array reader (Bio‐Rad).