Bio plex pro human cytokine 17 plex assay

The levels of IL-6 (Limit of detection (LOD): 2 pg/ml) and IL-8 (2 pg/ml) in cell culture supernatants from PMN were analyzed with ELISA plates from eBioscience (San Diego, CA, USA). In addition, levels of 17 cytokines were measured in supernatants from PBMC using Bio-Plex Pro Human Cytokine 17-plex Assay from Bio-Rad Laboratories (Herculeas, CA, USA) according to the manufacturer’s instructions. The following cytokines were measured: IL-1β (0.6 pg/ml), IL-2 (1.6 pg/ml), IL-4 (0.7 pg/ml), IL-5 (0.6 pg/ml), IL-6 (2.6 pg/ml), IL-7 (1.1 pg/ml), IL-8 (1.0 pg/ml), IL-10 (0.3 pg/ml), IL-12p70 (3.5 pg/ml), IL-13 (0.7 pg/ml), IL-17 (3.3 pg/ml), granulocyte-colony stimulating factor (G-CSF; 1.7 pg/ml), granulocyte monocyte-colony stimulating factor (GM-CSF; 2.2 pg/ml), interferon (IFN)-γ (6.4 pg/ml), monocyte chemotactic protein (MCP)-1 (1.1 pg/ml), macrophage inflammatory protein (MIP)-1β (2.4 pg/ml), and TNF-α (6.0 pg/ml). The cytokine levels were measured by the Bio-Plex 200 system (Bio-Rad).

The levels of CCL2, CCL4, CXCL8, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, TNF, and IFN-γ were measured using the Bio-Plex Pro™ Human Cytokine 17-plex Assay (Bio-Rad, Hercules, CA, USA), while CCL3, CCL5, and CXCL-10 were measured using the Bio-Plex Pro™ Human Cytokine 3-plex Assay (Bio-Rad), following the manufacturer’s instructions. Sample analysis was performed using the Bio-Plex™ 200 System (Bio-Rad) and Bio-Plex Manager™ software version 6.1 (Bio-Rad). However, IL-7 was not detectable, and CCL5 was detectable in only 42% of the total samples (or in only 31% of the pulmonary secretion supernatant); therefore, they were excluded from the analysis.

Cytokines in cell culture supernatants were analyzed by Bio-Plex Pro Human Cytokine 17-plex Assay (Bio-Rad Laboratories, Mississauga, Canada) according to the manufacturer's recommendation. Results were analyzed on a CS 1000 Autoplex Analyzer (Perkin Elmer Inc., Waltham, MA). TGFβ was detected by ELISA (R&D Systems).
IDO quantification
The enzyme IDO catalyzes the degradation of the essential amino acid L-tryptophan to N-formylkynurenine, which can be quantified in a colorimetric assay as described [66 (link)]. Briefly, supernatant was diluted with as solution of 30% trichloracetic acid (ratio 2:1), and incubated 30 minutes at 50°C. Samples were spin 1 minute at 15.400g and the supernatant was collected and diluted 1:1 with Ehrlich's reagent. Absorbance at 490 nm was detected with an ELISA plate reader and control wells values subtracted.

Briefly, 1.0 × 10⁵ FLS were cultured in 2 mL DMEM/10%FBS for 24 h and then washed twice in PBS. Adherent cells were treated with NDMVs in DMEM/10% FBS or with and without NDMVs at a V/C ratio of 100 for 6 h and 24 h, ±TNFα at 10 ng/mL. After incubation, cell medium was collected and ultra-centrifugated at 200,000× g for 60 min at 4 °C to remove any NDMVs or debris, and supernatants were retained. Cytokine/chemokine levels were measured using the Bio-Plex Pro Human Cytokine 17-plex Assay (Bio-Rad, Hercules, CA, USA), as per the manufacturer’s instructions (detectable range in Table S3). Briefly, 50 μL diluted magnetic beads was added to each well of an assay plate which was washed twice with Wash Buffer. An amount of 50 µL of culture supernatant was transferred to each well and incubated (with shaking) with beads for 30 min at RT. After washing the plate, 25 μL detection antibody was added and incubated (with shaking) for 30 min at RT. The plate was washed 3 times, and 50 μL diluted SA-PE was added and incubated for 10 min. Lastly, 125 μL assay buffer was added to resuspend the magnetic beads, and the plate was shaken for 30 s. The samples were then measured in a Bio-Plex 200 System (Bio-Rad, Hercules, CA, USA) alongside calibration standards.

A multiplex biometric immunoassay containing fluorescent-dyed microspheres conjugated with a monoclonal-specific antibody for a target protein was used for measurement of inflammatory mediators according to the manufacturer´s instructions (Bio-Plex Pro Human Cytokine 17-plex Assay; Bio-Rad Inc., Hercules, CA, USA). The mediators measured were: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17, G-CSF, GM-CSF, MCP-, MIP-1β, IFN-γ, and TNF mediator levels were determined by a multiplex assay reader from the Luminex Instrumentation System (Bio-Plex Workstation from Bio-Rad Laboratories, Inc.). Analyte concentration was estimated according to the standard curve using the Bio-Plex Manager software provided by the manufacturer. Values of unstimulated cultures were discounted from all stimuli.