Magpix milliplex kit

The serum levels of IL-6 and IFN- γ were determined using a MAGPIX Milliplex kit (Merck, Darmstadt, Germany), according to the manufacturer’s instructions. Briefly, 25 μL of serum samples, standards, and controls were added to a 96-well plate comprising an equal amount of assay buffer for serum samples or serum matrix for standards and controls. Magnetic beads coated with antibodies against the target cytokines were added to each well, and the plates were incubated on a plate shaker overnight at 4 °C. After washing with washing buffer in the kit, the samples were reacted with biotinylated detection antibodies for 1 h and then with streptavidin–phycoerythrin for 30 min. After washing and addition of loading buffer from the kit, the samples were analyzed by the MAGPIX system (Luminex, Austin, TX, USA). The results are presented in Supplemental Fig. S3.

Supernatant from THP-1 cells, incubated with non-biotinylated CMC-CNT, properdin-CMC-CNT, and MBP-TSR4+5-CMC-CNT for 24 and 48 h, were collected for measuring secreted cytokines, chemokines, growth factors, and other ligands and receptors. The analytes were measured using MagPix Milliplex kit (EMD Millipore) following the manufacturer’s protocol. 25 µL of assay buffer was added to each well of a 96-well plate, followed by addition of 25 µL of standard, control, or supernatant of THP-1 cells. 70 µL of a mixture of 36 individual capture magnetic beads was added to 3.5 mL of diluent buffer, vortexed, and 25 µL of the magnetic beads coupled to analytes were added to each well containing assay buffer, samples, and controls and incubated for 18 h at 4°C. The plate was washed with assay buffer, and 25 µL of detection antibodies (EMD Millipore) were incubated with the beads for 1 h at room temperature. 25 µL of streptavidin–phycoerythrin was then added to each well and incubated for 30 min at room temperature. Following the washing step, 150 µL of sheath fluid was added to each well, and the plate was read using the Luminex Magpix instrument. Assays were carried out in duplicate.

Measurement of cytokines and chemokines in serum samples was carried out as previously reported^{18 (link)}. The serum levels of IL-6, IP-10, MCP-1, and MIP-1β were determined using a MAGPIX Milliplex kit (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. Briefly, 25 μL serum samples, standards, and controls were added to a 96-well plate comprising an equal amount of assay buffer for serum samples or serum matrix for standards and controls. Next, magnetic beads coated with antibodies against the target cytokines were added to each well, and the plates were incubated on a plate shaker overnight at 4 °C. After washing with washing buffer in the kit, the samples were reacted with biotinylated detection antibodies for 1 h and then with streptavidin–phycoerythrin for 30 min. After washing and the addition of loading buffer from the kit, the samples were analyzed by the MAGPIX system (Luminex, Austin, TX, USA).

Supernatants were collected from the phagocytosis assay at 24 and 48 h to determine the levels of secreted cytokines (IL-6, IL-10, IL-12p40, IL-12p70, IL-1α, IL-1β, TNF-α, IL-13, IL-15, IL-17A, IL-9, and TNF-β), chemokines (MCP-3, MDC, Eotaxin, Fractalkine, GRO, IL-8, IP-10, MCP-1, and MIP-1α), growth factors (IL-2, EGF, FGF-2, G-CSF, GM-CSF, IL-3, IL-4, IL-5, IL-7, and VEGF), and other related ligands and receptors (IFN-α2, IFN-ϒ, FLT-3L, IL-1RA, and sCD40L). MagPix Milliplex kit (EMD Millipore) was used to measure immune response following the manufacturer’s protocol. 25 µl of assay buffer was added to each well of a 96-well plate, followed by the addition of 25 µl of standard, controls or supernatants of cells treated with M. bovis BCG in the presence or absence of properdin and MBP-TSR4+5. 25 µl of magnetic beads coupled to analytes of interest was added in each well and incubated for 18 h at 4°C. The 96-well plate was washed with the assay buffer, and 25 µl of detection antibodies was incubated with the beads for 1 h at room temperature. 25 µl of streptavidin–phycoerythrin was then added to each well and incubated for 30 min at room temperature with shaking at 750 rpm. Following a washing step, 150 µl of sheath fluid was added to each well, and the plate was read using the Luminex Magpix instrument. Assays were conducted in duplicate.

Concentrations of the following human cytokines and chemokines (CD40, EGF, ENA-78, FGF basic, G-CSF, GM-CSF, GROα, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-1 RII, IL-2, IL-3, IL-4, IL-5, IL-6ra, IL-6, IL-8, IL-10, IL-12 p70, IL-15, IL-17, IL-19, IL-27, IL-31, IP-10, MIP-1α, MIP-1β, TNF-α and VEGF) were measured by MagPix Milliplex kit (EMD Millipore, U.S.A). Magnetic beads coupled to specific analytes were incubated with the supernatant of myometrium cells treated with rhSP-A or rhSP-D at different time points and doses, and assay buffer. The samples were loaded on a 96 well plate and kept at 4°C for 18 h. After a series of washes, detection antibodies were incubated with the magnetic beads for 1 hour at room temperature and then with a Streptavidin-Phycoerythrin conjugate for 30 min. Finally, sheath fluid was added to each well and the plate was using the Luminex Magpix instrument, according to manufacturer’s instructions.

Interleukin-6 (sensitivity < 0.11 pg/mL), IL-8 (sensitivity < 0.13 pg/mL), IL-10 (sensitivity < 0.56 pg/mL), and GM-CSF (sensitivity < 0.35 pg/mL) concentrations in cell culture supernatant were determined using the MAGPIX Milliplex kit (Merck) following the manufacturer’s protocol. Untreated cells were taken as a negative control. Additionally, the MMP-9 concentration in culture supernatants was also measured using a sandwich ELISA (Sigma Aldrich, Merk) and read using a BT 2000 Microkinetics Reader at 450 nm. A standard curve was constructed to quantify the MMP-9 with a detection range of 8–6000 pg/mL.