Luminex 200 system

The media of in vitro polarised BMDM and B16F10 cell cultures (60–80% confluent) were changed for Opti-MEM, and the conditioned media (CM) harvested 24 h later. CM from ex vivo cultured alveolar macrophages were harvested 24 h after plating the macrophages in complete DMEM.
Blood was taken via cardiac puncture from euthanized mice bearing s.c. melanoma or lung metastases, and the sera were separated after letting the blood clot at room temperature for 30 min.
The CM and sera were cleared by centrifugation for 10 min at 12,000 RPM. The clear supernatants were subjected to Luminex assay using a Bioplex Mouse Group I 23-plex panel combined with single-plex components of IL-15, IL-18 and VEGF (BioRad, Watford, UK) on the Luminex 200 System according to the manufacturer’s protocol. Standard curves were optimised and protein concentrations calculated using the Bio-Plex Manager software v6.0 (BioRad) or Prism 8 (GraphPad, San Diego, USA).

We used multiplexed bead assay techniques for establishing the profiles of 39 immune mediator proteins that included cytokines, chemokines, and growth factors in serum and CSF. The selected panel was the most comprehensive available. Assay reagents and plates were obtained from well-validated commercial sources (Millipore®) [42 (link)]. The procedures followed recommendations and well-established protocols for evaluation of serum [42 (link), 43 (link)] and the CSF [44 (link), 45 (link)]. Only the first freeze-thaw aliquots were used for assay measurements. To achieve uniformity in the longitudinal assessment, assays for samples collected at different timepoints from the same individual were run simultaneously. Masked samples were measured in duplicates and blank values subtracted from all readings. Measurements and data analysis of all assays were performed with the Luminex-200® system in combination with Luminex manager software (Bioplex manager 5.0, Bio-Rad, Hercules, CA). We used standard operating procedures to guarantee the consistency, reproducibility, and reliability of the assays. Samples that exhibited unexpected or unacceptable variance (i.e., evidence of bead clumping, coefficients of variation greater than 20%, or unusual distributions of values) were re-tested.

Cytokine expression in serum was assessed by Bio-Rad BioPlex 200 instrument equipped with Bio-Plex Manager software version 6.0 (Bio-Rad Laboratories, Hercules, CA). The levels of interleukin-6 (IL-6), interleukin-1beta (IL-1β), interleukin-17 (IL-17), interferon gamma (IFN-γ), and TNF-α were detected using the Bio-Plex Pro Mouse Cytokine Th17 Panel A 6-Plex Group l Kit (Bio-Rad Laboratories,) on a Luminex 200 system (Bio-Rad Laboratories) according to the manufacturer’s instructions.

IL-12p40 and TNFα levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer’s protocol (U-Cytech, Utrecht, The Netherlands). Multiplex assays were performed using a customized non-human primate Milliplex Kit (Millipore, Billerica, MA). All cytokines, chemokines and growth factors were analyzed according to manufacturer’s protocol on a Luminex 200 system (Biorad).

Cytokines were measured in culture supernatant from unchallenged vaginal and rectal macaque explants after 24 hours of culture. A magnetic multiplex bead immunoassay (R&D Systems, Minneapolis, MN) was used to detect MCP‐1, MIP‐1β, RANTES, IP‐10, EGF, GM‐CSF, IFN‐γ, IL‐1β, IL‐1Ra, IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, IL‐15, IL‐17 and VEGF‐A on a Luminex 200 System (Bio‐Rad, Hercules, CA). Cytokine levels were normalized against total protein content as measured by a BCA protein assay (Bio‐Rad, Hercules, CA).

To determine the effects of CUR treatment on NESC and EESC, cytokine and chemokine levels were measured in conditioned media. Culture media were collected at 24 and 48 hr posttreatment of analysis of cytokines (tumor necrosis factor‐α [TNF‐α], vascular permeability factor/vascular endothelial growth factor [VEGF], platelet‐derived growth factor [PDGF], interferon γ [IFNγ], fibroblast growth factors [FGF], interleukin [IL]‐1b (IL‐1β), IL‐1a (IL‐1α), IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐8, IL‐9, IL‐10, IL‐12, IL‐13, IL‐15, IL‐17) and chemokine (eotaxin [CCL11], granulocyte‐colony stimulating factor [G‐CSF], granulocyte‐macrophage colony stimulating factor [GM‐CSF], IFNγ‐induced protein 10 [IP‐10/CXCL10], MCP‐1/CCL2, macrophage inflammatory proteins 1a [MIP‐1α/CCL3], MIP‐1β/CCL4, RANTES [CCL5]) using Bio‐Plex Pro Human Cytokine, Chemokine, and Growth Factor Magnetic Bead‐Based Assays (BioRad, Hercules, CA) coupled with the Luminex 200 system (Austin, TX) according to the manufacturer’s protocol. Samples were tested at a 1:2 dilution using optimal concentrations of standards and antibodies according to the manufacturer’s protocol.