Xponent software package

A total of 48 cytokines including interleukins, chemokines, colony stimulating factors, growth factors, interferons, growth factors and tumor necrosis factor were measured in this study. Cytokine concentrations were measured in serum and human brain tissue TBS extracts using the Pro Human Cytokine Screening Panel 48-plex assay (Bio-Rad, Hercules, California, USA) and 5-point standard curve consisting of S3, S4, S5, S6, S7 standards and a blank, which covered our sample concentration range. Briefly, samples were diluted (serum 1:4, brain tissue 1:2), in sample diluent, and incubated with detection antibodies coupled to magnetic beads, washed using a Bio-Plex Pro wash station and incubated in streptavidin–phycoerythrin before wells were quantified using a Xponent software package (Luminex, Austin, TX). Provided standards generated a five-parameter standard curve for all 48 cytokines and unknown concentrations were calculated with Bio-Plex Manager software 6.1. The intra-assay %CV for the serum plate was 2.68–4.97 (average: 3.51) and for the brain tissue plate 3.65–6.42 (average: 4.86). The inter-assay %CV for the two plates was 4.48–13.57 (average: 7.92).

IL-6 was measured using the magnetic human cytokine ELISA assay (Bio-Rad) following the manufacturer’s instructions. Plates containing the serum samples and standards were analyzed using Magpix plate reader (Luminex), and the concentration of IL-6 was determined using the Xponent software package (Luminex). C3 was measured by western blotting (see below). Calcium assay was carried out following the manufacturer’s protocol (Abcam, ab102505). Briefly, 50 µl of samples and standards were added to 96-well plates. Then, 90 µl of the chromogenic reagent was added to each well, followed by 60 µl of calcium assay buffer. The plates were incubated at room temperature for 10 min in the dark and read in a microplate reader at 575 nm.

The human serum was separated from peripheral blood of the individuals with a known genotype. Peripheral blood (3 mL) was collected during regular control medical examination and inclusion criteria were that patients should be in the stable state of the disease. Blood samples were centrifuged and serum was separated and stored at −80 °C.
Concentrations of the selected cytokines in the sera of COPD patients and healthy donors were measured using a ProcartaPlex High Sensitivity Assay, with a corresponding bead set (Thermo Fisher Scientific, Waltman, MA, USA), according to manufacturer’s recommendation. Following the detailed protocol which is described here [21 (link)], labeled samples were analyzed by use of a Luminex 200 instrument. The concentration of tested cytokines was determined by interpolation from a standard curve using the xPONENT software package (Luminex, Austin, TX, USA).

Concentrations of IL-1β in EDTA plasma samples obtained from patients with COPD and healthy individuals were measured using a ProcartaPlex High Sensitivity Assay, with a corresponding IL-1β bead set (Thermo Fisher Scientific, Waltman, MA, USA), according to the manufacturer’s recommendations. Briefly, 50 μL of antibody-coated magnetic beads were added per well into a 96-well plate and washed. Afterwards, 25 μL of samples or standards were added to a 25 μL universal assay buffer, and the plate was incubated for 30 min at room temperature (RT) and overnight at 4°C, with shaking. After the washing steps, 25 μL of detection antibodies were added to the wells and the plate was incubated for 30 min at RT, with shaking. After the washing, 50 μL of a streptavidin-phycoerythrin conjugate was added to the wells. After the incubation and washing steps, 50 μL of amplification reagent 1 was added to the wells, and the plate was incubated for 30 min at RT, with shaking. Then, amplification reagent 2 (50 μL) was added to the wells, and, following the incubation and washing steps, the beads were resuspended in 120 μL of reading buffer and analyzed by a Luminex 200 instrument (Luminex Corporation, Austin, TX, USA). The concentration of IL-1β was determined by interpolation from a standard curve using the xPONENT software package (Luminex Corporation, Austin, TX, USA).

A magnetic human cytokine multiplex assay (Biorad) was used to simultaneously measure 27 cytokines (MIP1β, IL-6, IFNγ, IL-1RA, IL-5, GM-CSF, TNFα, CCL5, IL-2, IL-1β, CCL11, bFGF, VEGF, PDGF, CXCL10, IL-13, IL-4, MCP-1, IL-8, MIP1α, IL-10, GCSF, IL-15, IL-7, IL-12(p70), IL-17A, and IL-9) in the serum samples. The bioplex assay was performed exactly as per the manufacturers’ instructions, using the recommended one in four dilution of serum, and plates analyzed using a Magpix plate reader (Luminex). Enclosed standards were used to generate an eight-point standard curve to which a five-parameter logistic curve was fitted and used to quantify unknown cytokine concentrations using the Xponent software package (Luminex). Reference pool standards were included in every plate. The coefficient of variance between duplicates was generally <10%. The coefficient of variance between reference pool standards run on separate plates was ~10–40%, depending on the cytokine of interest. Once the ELISA assays were completed, the raw data were uploaded to the Michael J Fox Foundation LCC website and the investigators were then unblinded to facilitate analysis.