Procartaplex kit

CD4⁺CD49b⁺LAG3⁺ Tr1 cells from either vehicle or vaccine-treated NOD mice were cultured with CD3/CD28 latex beads and either 50 μg/ml of insulin peptide B9-23(SHLVEALYLVCGERG, CAT# AS-61532, Anaspec), 50μg/ml of GAD65 (SRLSKVAPVIKARMMEYGTT, p524-543, CAT#AS-62757, Anaspec), or 50 μg/ml of OVA peptide (ISQAVHAAHAEINEAGR, CAT# 323-339, GenScript), and supernatants were collected after 72 h. Mouse IL10 levels in the supernatants and serum circulating levels of IL10 and TGFβ were quantified using the Mouse Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.
To assess antigen-specificity, splenocytes (5 × 10⁵ cells) obtained from different treated groups of mice followed by in vitro stimulation by culturing with insulin peptide B9-23 for 72 h. The levels of IFNγ, TNFα, IL12p70, and IL17A were quantified in cell-free supernatants using a ProcartaPlex kit (eBioscience) and Bio-Plex analyzer (Bio-Rad, Hercules, CA).

The cytokine levels in mice plasma were measured by multiplex immunoassay with ProcartaPlex Kit (ThermoFisher Scientific, MA). In total, 11 cytokines were tested: interleukin-4 (IL-4), IL-5, IL-6, IL-1β, IL-12, interferon-α (IFNα), IFNγ, tumor necrosis factor-α (TNFα), granulocyte macrophage colony stimulating factor (GM-CSF), transforming growth factor β (TGFβ), and vascular endothelial growth factor (VEGF).

Serum cytokines and chemokines, fractalkine (CX3CL1), gamma interferon (IFN-γ), IFN-α, interleukin-12 p70 (IL-12p70), IL-13, IL-1b, IL-6, IL-8, IL-18, IL-2, IL-10, tumor necrosis factor α (TNF-α), monocyte chemoattractant protein 1 (MCP-1), monokine induced by IFN-γ (MIG), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, IFN-γ-inducible 10-kDa protein (IP10), and the stromal cell-derived factor 1α (SDF-1α) were analyzed using a multiplex Luminex assay (ProcartaPlex kit; ThermoFisher, Waltham, MA, USA) according to the manufacturer’s recommendations. Serum samples from healthy residents (n = 51) of similar ages and sex from the same region were tested simultaneously. All samples were stored at –40°C and tested in batches in May 2017.

ProcartaPlex kit (Thermo Fisher, USA) was used to measure the concentrations of cytokines/chemokines in the culture supernatants of primary astrocytes and microglia and homogenates of mouse striatum according to the manufacturer’s instructions. Briefly, 50 μl cell culture supernatants or 25 μl tissue homogenates were added into each filter plate well containing conjugated antibody beads, then the plate was incubated at room temperature for 30 min at 500 rpm and 4°C overnight. After washing, the beads were incubated with detection antibody and subsequently SA-PE at 500 rpm for 30 min, respectively, at room temperature. The beads were re-suspended in reading buffer and then the signals were detected by Luminex 200 (Luminex, USA).

Plasma aliquots were thawed and prepared following kit manufacturer guidelines. All samples were analyzed in duplicate. MCP-1, soluble (s)CD163, IP-10, galectin-1, galectin-3, galectin-9, IL-6, TNF-α, TNFR1, D-dimer, and cystatin C were measured using custom Luminex kits from (R&D Systems) and CRP was measured using the Procartaplex kit (Thermofisher). Data was acquired on a Luminex 200^TM analyzer (Luminex) and analyzed using MILLIPLEX^® Analyst software (Millipore). Neopterin and sCD14 were measured via ELISA (Neopterin competitive enzyme immunoassay, ALPCO, NH, USA; Human CD14 Quantikine ELISA kit, R&D Systems). Optical density was read with a microplate spectrophotometer (Bio-Rad) and data analysis, including four parameter logistic standard interpolation, was carried out using the online MyAssays Ltd. data analysis tool.

The cytokine assays were performed on serum from PB samples collected from patients at multiple time points after CAR-NK cell infusion using the Procartaplex kit from Thermo Fisher following the manufacturer’s instructions. The qPCR assays were performed on serial PB samples as previously described^{13 (link)}.