Milliplex analyst

Cytokine profiling of samples were performed using MILLIPLEX MAP mouse cytokine/chemokine magnetic bead panels (MCYTMAG-70K-PX32) based on the Luminex xMAP technology (Millipore Corp., Billierica, MA) using standards and controls for each cytokine and chemokine provided by the manufacturer. The cytokine panel includes VEGF, Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC-like, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES and TNF-α. Concentrations are reported in pg/ml. Assays were performed with lung homogenates as per manufacturer’s instructions using at least five biological and two technical replicates. Data acquisition was done using xPonent and Milliplex analyst (Millipore Corp., Billierica, MA ). Sample wells with less than 20 pg/ml were invalidated. Exploratory data analyses enabled logarithmic base 2 transformations for all continuous variables. Linear regression analysis was used to test the comparisons between TIGR4 and ΔpotABCD. Sensitivity analysis was done using non-parametrics methods and found similar qualitative results. Stata 13.1 (Stata Corporation, College Station, TX) was used to conduct statistical analysis. The significance level was set at 0.05.

PBMCs were resuspended at a concentration of 2 × 10⁵ cells per well in a 96-well U-bottom plate (Corning) in 200 µL RPMI-1640 media (Gibco) supplemented with 10% autologous PPP, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. PBMCs were incubated for 24 h at 37 °C in a humidified incubator at 5% CO₂ with indicated treatments. After culture, plates were centrifuged at 500 g and supernatants were removed by pipetting without disturbing the cell pellet. Cytokine production was measured in cell culture supernatants using customized Milliplex human cytokine magnetic bead panels (Milliplex). Assays were analyzed on the Luminex FLEXMAP 3D employing xPONENT software (Luminex) and Millipore Milliplex Analyst. Cytokine measurements were excluded from analysis if <30 beads were recovered. CCL2 and CCL3 were measured by ELISA kits (Invitrogen).

At day 20, tumors were harvested and weighed. Tumor samples (5 μl/mg) were lysed in 20% Cell Lysis Buffer with PMSF (Cell Signaling Technology) and supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Each tumor was homogenized in bead beater tubes, and the lysate was stored at -80°C. The concentration of 32 cytokines and chemokines in the tumor lysates (MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel, Millipore) were determined by a multiplex immunoassay following manufacturer’s instructions. The MAGPIX System (Millipore) was used to read the multiplex plate. Concentrations were determined using a standard curve and their respective median fluorescence intensity (MFI) readings (Milliplex Analyst, Millipore). The data underwent log and Z-transformation followed by unbiased hierarchical clustering using Matlab R2019.