Luminex magpix

The levels of cytokines were measured by multiplex magnetic bead immunoassay system, (Milliplex MAP kit, Merck Millipore, Molsheim, France), in BAL supernatants that had been previously concentrated 5× using Amicon Ultra-centrifugal filters, (Merck Millipore). Mouse 17-plex cytokine/chemokine detection kit (G-CSF, M-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-10, IL-13, CXCL10/IP-10, CXCL1/KC, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, and TNF) was used for cytokine quantification in BAL supernatants of naïve and NA-injected mice, whereas Mouse 8-plex cytokine/chemokine detection kit (Milliplex MAP kit) (IL-1α, IL-1β, IL-6, IL-10, IL-13, CXCL10/IP-10, CXCL1/KC, and CCL2/MCP-1) was used for cytokine quantification in BAL supernatants of mice administered with PBS- or CL-containing liposomes. Beads were analyzed with Luminex MAGPIX, (Merck Millipore). The detection limits for these cytokines was 3.2 pg mL⁻¹.

Monocyte samples (white blood cells) were used to evaluate complex I-V function in a multiplex ELISA assay. Cells were lysed with phosphatase inhibitors, protease inhibitors and OXPHOX lysis buffer before being centrifuged at 14000 g for 20 minutes at 4 C. 96-well plates were activated with 200 µL of wash buffer for 10 minutes. 25 µL of sample and 25 µL of magnetic beads were added to each well and incubated for 2 hours at room temperature. Plates were washed and 50 µL of detection antibody was added to each well and incubated for an hour at room temperature. Plates were washed and 50 µL of streptavidin-phycoerythrin was added and incubated with samples for 30 minutes. Plates were washed, and 100 µL of drive fluid was added to the wells. The plates were read using Luminex Magpix (EMD Millipore) software, and analysis was performed with xPONENT software. Complex function is reported as a percentage against each subject’s individual nicotinamide nucleotide transhydrogenase levels (%NNT); a nucleus-encoded protein present in the inner mitochondrial membrane that is closely related to mitochondrial oxidative phosphorylation^{58 (link)}.

Twenty-four and forty-eight hours after cell plating, SNs were collected from Treg and ASC monocultures, as well as direct and indirect cocultures. SNs were frozen and stored at −80 °C until analysis. Concentrations of 48 mediators: sCD40L, EGF, eotaxin, FGF-2, FLT-3L, fractalkine, G-CSF, GM-CSF, GROα, IFNα2, IFNγ, IL-1α, IL-1β, IL-1RA (IL-1 receptor antagonist), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10, MCP-1/CCL2, MCP-3, M-CSF, MDC/CCL22, MIG/CXCL9 (monokine induced by IFNγ), MIP-1α/CCL3, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES/CCL5, TGFα, TNFα, TNFβ and VEGF-A were measured with Human Cytokine/Chemokine/Growth Factor Panel A 48 Plex Kit (Merck) and analysed with Luminex (MAGPIX, Merck Millipore) according to the manufacturer instructions. In addition levels of, IL21 and IL35 were measured with ELISAs (BioLegend and Elabscience, respectively).

The total IgE level was determined in the mouse serum using MILLIPLEX^® MAP Mouse IgE Single Plex Magnetic Bead Kit (Merck Millipore, Banglore, India) as per the manufacturer’s protocol and the samples were analyzed using the Luminex Magpix (Merck Millipore, Banglore, India) [22 (link)]. The total IgG level in the mouse serum was measured using the Amplex ELISA development kit (San Francisco, CA, USA) for mouse IgG as per the manufacturer’s protocol. All the samples were analyzed at 405 nm using ELISA plate reader. The results obtained were means of duplicate determination with variation less than 10%. Further, the IgE and IgG concentrations were determined by comparing the mean OD values of the tested sera with the mean OD values of the standard. IgE and IgG titers were calculated by multiplying the dilution factor of the test sera and expressed in ng/mL.

Viruses in supernatant samples are inactivated with 0.5% Triton X-100 for 1 hour at room temperature. The levels of immune mediators, including cytokines, chemokines, and growth factors were quantified using the Milliplex Human Cytokine/Chemokine/Growth Factor Panel A (Merck Millipore, USA) following the manufacturer’s instructions. This system allowed quantitative measurements for 25 biomarkers: FGF-2, G-CSF, GM-CSF, IFN-α2, IFN- γ, IL-1 β, IL-1RA, IL- 2, IL-3, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 p70, IL-17A, IL-22, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-AA, PDGF-BB, TNF-α, and VEGF-A. The analysis was performed on a Luminex MAGPIX (Luminex Multiplexing Instrument, Merck Millipore) with a minimum of 50 beads collected per analyte per well. Belysa software was used to generate standard curves for each analyte using five parametric logistic fit models. Statistical data analysis was performed by using the one-way ANOVA Kruskal Wallis test with Dunn’s multiple comparison test and figures were generated using GraphPad Prism 8.0 (GraphPad Software Inc, USA).