Bio plex 200 suspension array system

Plasmas from lupus mice with different treatments were harvested and stored at −80°C prior to be subjected to a multiplex immunoassay. Using a Milliplex MAP assay kit (Merck Millipore, Billerica, MA, USA), several cytokines (i.e., TGF‐β, IL‐10, IL‐1β, MIG, IL‐12, IFN‐γ, TNF‐α, IL‐17A and IL‐6) were measured through a Bio‐Plex 200 suspension array system (Bio‐Rad Laboratories, Hercules, CA, USA).

After 24-h incubation with samples in 96-well plates, the following cytokines production from each well containing 10,000 cells was analyzed: interleukin (IL)-6; IL-10, tumor necrosis factor (TNF)-α; granulocyte colony-stimulating factor (G-CSF); granulocyte macrophage colony-stimulating factor (GM-CSF); lipopolysaccharide-induced CXC chemokine (LIX; CXCL5); monocyte chemotactic activating factor (MCP)-1; macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, RANTES (CCL5; regulated on activation, normal T cell expressed and secreted), vascular endothelial growth factor (VEGF); interferon gamma-induced protein 10 (IP-10; CXCL10); and leukemia inhibitory factor (LIF). Cytokines were measured using a Luminex assay based on xMAP technology with multiplex cytokine assay kits and a Bio-Plex 200 suspension array system (Bio-Rad, Hercules, CA, USA), as described previously [4 (link),5 (link),6 (link),7 (link),8 (link),9 (link)]. The assay used in this experiment was designed for the multiplexed quantitative measurement of multiple cytokines in a single well, using as little as 50 µL of cell culture supernatant. Baicalein, a well-known anti-inflammatory flavonoid, was used as a positive control.

Proinflammatory cytokine and chemokine levels in the CSF and plasma were measured during the acute phase of disease and before the start of treatment with antibiotics. Analysis was performed with microsphere-based multiplex assays (xMAP Luminex technology) using the Bio-Plex 200 suspension array system (Bio-Rad, Hercules, CA, USA). A human cytokine Lincoplex Kit (HCYTO-60 k, Millipore, Billerica, Ma, USA) was used to analyze a set of 12 cytokines and chemokines (TNF-α, IL-6, IL-1β, INF-γ, IL-2, IL-10, IL-1RA, MIP-1α/CCL3, MIP-1β/CCL4, MCP-1/CCL2, G-CSF and IL-8/CXCL8). The samples were processed and measured according to the manufacturer’s instructions, as described previously [10 (link), 12 (link), 13 (link)]. Cyto/chemokine expression was measured in duplicate, and the inflammatory modulators levels were analyzed in relation to a parametric logistic curve using Bio-Plex manager 4.01 software and expressed as pg/ml. Due to the low quantities of biological material, evaluation of all patients was not possible.

Concentrations of the gonadotropins luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) were assessed using the Milliplex Map Pituitary Magnetic Bead Panel Kit (PPTMAG‐86K; MilliporeSigma, Burlington, MA, USA) inter‐assay cv <20%, intra‐assay cv <15%, range 24.4‐100 000 pg/mL for FSH and 4.9‐20 000 pg/mL for LH according to the manufacturer's instructions. A Bio‐Plex 200 suspension array system was used to measure plasma concentrations and data were analyzed with Bio‐Plex Manager software (Bio‐Rad Laboratories, Hercules, CA, USA). Our experimental intra CV for FSH assay <6.5 and for LH assay <3.4.
The steroids in mouse plasma and testis lysate were measured using an isotope‐dilution TurboFlow liquid chromatography‐tandem mass spectrometry method as previously described.¹⁴, ¹⁶ For progesterone, 17‐OH‐progesterone (17‐OHP), androstenedione, and testosterone the limits of quantification were 0.036 nM, 0.1 pM, 0.012 nM, and 0.042 nM, respectively, and the interday variation, expressed as the relative standard deviation for these analytes were testosterone was ≤5.3% and ≤4.2% for low and high spike levels of control materials, respectively, included three times each in every analytical batch. Chemical analyses were performed at the Dept. of Growth and Reproduction, Rigshospitalet, Copenhagen University Hospital.

Procartaplex multiplex Immunoassay kit was purchased from Thermo Fisher Scientific Waltham, MA, USA to determine and quantify released cytokines in cell culture supernatants. Concentration of IL-6, IL-8, IL-1α, TNF-α protein in the cell culture supernatant were measured after treatment of fpEC with oxysterols according to the user’s manual. The BioPlex-200 suspension array system was used to measure the fluorescence intensity of the samples (Biorad, Hercules, CA, USA). All obtained cytokine concentrations in the supernatant were normalized to total cell protein concentration, which was determined by BCA protein quantification assay.

FBMFs were cultured in DMEM with 10% FBS and 1% streptomycin/penicillin at a density of 2 × 10⁵ cells/10 cm dish until confluence for 5 days at 37°C incubation with 5% CO₂, and then the CM was collected and centrifuged at 1000 rpm for 5 min and frozen at −20°C until needed (not exceeding 1 week). For the control group, DMEM with 10% FBS was used. The cytokine profiles in the CM of fBMF cells were analysed using a Bio‐Plex Pro‐human cytokine 27‐plex assay (Bio‐Rad Laboratories) in accordance with the manufacturer's instructions. In brief, 100 μl of assay buffer and 50 μL of beads were added to the assay plate. The standard (27 types of cytokines) was reconstituted and diluted in a fourfold dilution series. The samples (50 μl) were added to each well and rinsed twice with wash buffer. After 1 h of shaking in the dark, the wells were rinsed three times with wash buffer before the detection antibody was added to each well. After another washing step, 50 μl of streptavidin‐phycoerythrin solution was added to each well, and they were incubated for 10 min. After the last incubation step, the beads were resuspended in 125 μl of assay buffer with shaking at 1100 rpm for 30 s. The cytokine data were analysed using the Bio‐Plex 200 suspension array system (Bio‐Rad Laboratories). The cytokine protein expression of fBMF was compared with the standard DMEM with 10% FBS (control group).