Xponent software

Retinas were sonicated and run in singlet on the Milliplex MAP Mouse Cytokine/Chemokine Magnetic Bead Panel I 25-plex assay per manufacturer’s protocol (EMD Millipore, Billerica, MA, United States). Plates were read on a Luminex MAGPIX with xPONENT software (Thermo Fisher Scientific, Waltham, MA, United States) using settings stated in the manufacturer’s protocol. Multiplex ELISA plates were read and analyzed through the use of the Vanderbilt Hormone Assay and Analytical Services Core.

The serum levels of soluble CD40 ligand (sCD40L), interferon (IFN)-α2, IFN-γ, IL- interleukin-1 receptor antagonist (1RA), IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-18, tumor necrosis factor alpha (TNF-α), Angiopoietin-2, Endoglin, Endothelin-1, soluble endothelial selectin (sE-Selectin), Thrombomodulin, soluble receptor for advanced glycation end-products (s-RAGE), soluble Intercellular Adhesion Molecule 1 (sICAM-1), P-Selectin, soluble vascular cell adhesion molecule 1 (sVCAM-1), and Plasminogen activator inhibitor-1 (PAI-1) were determined with the bead-based multiplex immunoassay Milliplex Map (EMD Millipore, Burlington, MA, USA). Samples were processed following the manufacturer’s instructions, and data were analyzed by the MAGPIX system provided with the xPONENT Software (Luminex, ThermoFisher Scientific, Waltham, MA, USA). The von Willebrand factor (vWF) was measured with a commercial ELISA kit (Thermofisher, Waltham, MA, USA) following the manufacturers’ instructions.

Retinas were sonicated and run in singlet on the Milliplex MAP Mouse Cytokine/Chemokine Magnetic Bead Panel I 25-plex assay per manufacturer’s protocol (EMD Millipore, Billerica, MA). Plates were read on a Luminex MAGPIX with xPONENT software (Thermo Fisher Scientific, Waltham, MA) using settings stated in the manufacturer’s protocol. Multiplex ELISA plates were read and analyzed through the use of the Vanderbilt Hormone Assay and Analytical Services Core.

To measure cytokines and chemokines produced by BAL AM, cells suspended in complete RPMI containing 2% FBS were plated in a 96-well flat bottom plate at 100,000 cells per well and rested for 3 h. After washing cells, fresh complete RPMI containing 2% FBS with and without 1.6 μg ml⁻¹ of M. tuberculosis lysates was added to wells and incubated for 12–14 h at 37 °C and 5% CO₂. Collected culture supernatants were frozen at −80 °C until measurement of protein levels. Whole blood was diluted with an equal volume of RPMI and 400 µl was plated in a 48-well flat bottom plate and incubated for 12–14 h at 37 °C and 5% CO₂ with or without stimulation with 1.6 µg ml⁻¹ of M. tuberculosis lysates. Plasma was then collected and stored at −80 °C until the measurement of protein levels. Cytokines/chemokine levels were quantified by using MCYTOMAG-70K mouse chemokine and cytokine detection kit (Millipore Sigma, Oakville, ON) according to the manufacturer’s instructions. Plates were read on a MagPix reader and concentrations of cytokines/chemokines were determined by using xPONENT software (Thermo Fisher Scientific).