Bio plex pro mouse cytokine 23 plex panel

At 17 days after disease induction (10 days after DC transfer), CD4⁺ T cells (3 × 10⁵ per well of a 96-well plate) isolated from EAU model mice by MACS were cultured for 24 h in 200 μl of RPMI 1640 medium. The concentrations of cytokines in the culture supernatants were then measured with the use of a Bio-Plex Pro Mouse Cytokine 23-Plex Panel and Bio-Plex Manager software version 4.1.1 (Bio-Rad, Hercules, CA).

At the time of sacrifice, blood was drawn by cardiac puncture. Levels of 25(OH)D₃ were measured in the serum by liquid chromatography tandem mass spectroscopy (LC/MS/MS) [24 (link)]. Levels of 1,25(OH)₂D₃ were measured using IDS EIA ELISA kits (Immunodiagnostic Systems, Fountain Hills, AZ, USA) as described by the manufacturer.
Serum cytokines were measured using Bio-Plex Pro™ Mouse Cytokine 23-plex panel (Bio-Rad Laboratories, Hercules, CA, USA) as per the manufacturer’s instructions. The cytokines analysed included interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40, IL-12 p70, IL-13, IL-17, eotaxin (CCL11), G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α.

Inflammatory mediators in BAL were analysed for the presence of 23 cytokines and chemokines (Bio-Plex™ Pro Mouse Cytokine 23-plex panel, BioRad, Hercules, CA, USA) (Table 
1). The analysis was performed in duplicates on a Bio-Plex™ system (Luminex Bio-Plex™ 200 System, Bio-Rad) according to the manufacturer’s instructions.

Eyeballs removed 7 days after CNV induction (10 laser spots per eye) were trimmed of extraocular muscles and soft tissue. The retina and choroid were isolated and were separately pooled (n = 6 to 10) to reduce biological variability. The tissue was homogenized in 0.1 ml per eye of a lysis buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN), and the homogenate was centrifuged at 16,200 × g for 10 min at 4°C. The protein concentration of the resulting supernatant was determined with a DC (detergent-compatible) protein assay (Bio-Rad, Hercules, CA), and the supernatant was then stored at –80°C until assay of cytokine and chemokine concentrations with the use of a Bio-Plex Pro Mouse Cytokine 23-Plex Panel and Bio-Plex Manager software version 4.1.1 (Bio-Rad).

Serum cytokine concentration was determined by a 23-plex assay kit (Bio-Plex Pro Mouse Cytokine 23-Plex Panel, Bio-Rad, Hercules, CA, United States) and analyzed using MAGPIX system (Luminex Corporation) and Bio-Plex Manager 6.1 software (Bio-Rad) according to the manufacturer’s instructions. The kit assessed the following cytokines and chemokines: IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1a, MIP-1b, RANTES, and TNF-α.

A 23-plex assay kit (Bio-Plex Pro Mouse Cytokine 23-Plex Panel, Bio-Rad, Hercules, CA, United States) was used to detect the concentration of serum cytokines following the manufacturer’s instructions. The data were further analyzed using a MAGPIX system (Luminex Corporation, Austin, TX, United States) and the Bio-Plex Manager 6.1 software (Bio-Rad, Hercules, CA, United States).