Milliplex map human cytokine chemokine magnetic bead panel

To determine the effect of KDG perturbation on cytokine expression, experiments were carried out as above with three separate donors and two independent experiments per donor. Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel (38plex from Millipore) assays were set up according to standard procedures using undiluted and 5× diluted supernatants. All out-of-range values were removed, and, for any cytokine, if fewer than 60% of the samples were within range, no analysis was performed. Cytokine response was scaled by dividing over the average for the non-target control by treatment (LPS⁺ or LPS⁻), donor and experiment, or plate. The difference (fold change) in cytokine concentration relative to the non-target control for each siRNA was tested using linear mixed modeling. Fixed effects consisted of a categorical variable for siRNA, and random effects reflected the replicates within each donor and experiment in the first data set and the replicates within each plate in the second.

The v7D-, vOka-, or mock-cell-lysate-pulsed DCs were co-cultured with autologous CD4⁺ or CD8⁺ T cells, isolated using Dynabeads™ FlowComp™ Human CD4/CD8 Kit (Thermo Fisher Scientific) from autologous PBMCs, at a ratio of 1:10. The T-cell concentration was 2 × 10⁵/mL. The stimulations were done in complete RPMI 1640 medium with 10% FBS over a period of 5 days. T-cell proliferation was examined daily using the 5-bromo-2′-deoxyuridine (BrdU) ELISA assay (Sigma) and by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling (Thermo Fisher Scientific) after five days of co-culture. The IFN-γ ELISPOT assay (Mabtech) was performed on day 5 after co-culture per the manufacturer’s instructions. Cytokines/chemokine production in the DC-T-cell co-culture supernatants was determined after 5 days of co-culture using the Millipore MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel.

Whole blood was collected by venipuncture at the enrollment visit. Blood for cytokine analysis was collected into serum separator tubes and centrifuged and aliquoted into cryovials following a standard protocol during or immediately following the study visit. Serum aliquots were frozen and stored at -80°C pending shipment to the biorepository and subsequently to the analyzing laboratory. As freeze-thaw cycles can alter cytokine measures, samples in our analysis were not freeze-thawed[18 (link)]. Total numbers of eosinophils and neutrophils were determined by the product of the differential and total cell count. Using 25 μL of participant serum, cytokines were quantified according to the manufacturer’s instructions using a premixed 38 Plex MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel (Millipore Corporation, Billerica, MA) and analyzed on a Luminex 100/200 analyzer (BioRad, Hercules, CA). Serum cytokine concentrations were determined using Bio-plex 6.1 software (BioRad, Hercules, CA). A similar approach for analyzing cytokines in BAL (bronchoalveolar lavage) fluid from asthmatics has been used previously[19 (link)]. Cytokines included in the analysis are listed in S1 Table.

After 12-hour overnight fasting, 40 mL of blood was extracted into vacutainer tubes and stored for subsequent analysis of fasting glucose and insulin, blood lipids, total cholesterol, triglycerides, anti- and pro-inflammatory biomarkers, and angiogenesis markers.
IFN-γ, IL-10, IL-12p70, IL-1ra, IL-1β, IL-6, TNF-α, VEGF and PDGF were analyzed using Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel (Millipore Corp., Billerica, MA), following the manufacturer’s instructions.

Routine biochemical (creatinine, potassium, sodium, albumin, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), vitamin B12 and serum folate test) and hematological tests (complete blood count, iron, transferrin saturation index (TSI), ferritin) were performed. To evaluate micronutrients, magnesium, cooper, phosphate, zinc, calcium and 25-OH vitamin D were also determined.
Finally, interleukins IL-5, IL-12 and IL-8 were determined obtaining 5 ml of peripheral blood by venipuncture into EDTA-K3 tubes. Plasma was separated by centrifugation and frozen at -20°C until use. Samples were prepared for analysis in a 96-well plate utilizing a custom 3-cytokine Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel (Millipore Corp., Billerica, MA) following the kit-specific protocols provided by Millipore. Interleukins were simultaneously quantified using the analytical test instrument Luminex-200, which utilizes xMAP technology (Luminex Corp., Austin, TX), and xPONENT3.1 software (Luminex). The intra-assay coefficient of variation was 7% and the interassay was 11%. The values were presented in picograms per milliliter (pg/mL).

CD14⁺ monocytes were isolated from PBMCs (Hemacare) with positive selection using Dynabeads™ FlowComp™ Human CD14 Kit according to manufacturer’s instructions (Thermo Fisher Scientific). Then, monocytes were seeded in six-well plates at a density of 2 × 10⁶ cells per well and cultured at 37 °C/5% CO₂ for 6 days in complete RPMI 1640 medium containing 10% FBS, 100 ng/mL GM-CSF (R&D system), and 40 ng/mL IL-4 (R&D system) to generate monocyte-derived immature DCs (iDCs). To generate v7D- or vOka-pulsed DCs, iDCs were seeded in 6-well plates at a density of 5 × 10⁵ cells per well and incubated with cell-free viruses of v7D or vOka (MOI = 0.01), or mock-infected MRC-5 cell lysate as a negative control, at 37 °C/5% CO₂ over a period of 5 days. LDH release (Thermo Fisher Scientific) and cytokines/chemokine analysis (Millipore, MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel) in the supernatants during co-incubation were performed daily per the manufacturer’s instructions. GM-CSF and IL-4 were maintained in a culture medium during all DC experiments.