Xponent 4

The following 48 analytes were measured at a protein level by multiplex xMAP technology using the MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel A kit (HCYTA-60K-PX48, Merck, Darmstadt, Germany): sCD40L, EGF, Eotaxin-1/CCL11, FGF-2/FGF-basic, Flt-3 ligand, Fractalkine/CX3CL1, G-CSF, GM-CSF, GROα, IFNα2, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17A/CTLA8, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10/CXCL10, MCP-1/CCL2, MCP-3/CCL7, M-CSF, MDC/CCL22, MIG/CXCL9, MIP-1α/CCL3, MIP-1β/CCL4, PDGF-AA, PDGF-AB/BB, RANTES/CCL5, TGF-α, TNF-α, TNF-β, and VEGF-A. Multiplex-based assay read-out was performed using MAGPIX system (Merck) with the xPONENT 4.2 software (Merck) in accordance with the manufacturer’s instruction with overnight incubation of the samples with primary antibodies. Final analysis was carried out with the MILLIPLEX Analyst v5.1 software (Merck). Measurements were performed twice for each sample. Release of the analytes in control and experimental samples was compared with unpaired two-sample t-test using GraphPad Prism v.8.0.1 (GraphPad Software, Inc, San Diego, CA, USA). The p values ≤ 0.05 were considered significant.

The cell lysates were collected and the level of phospho-protein expression was analyzed with the MagPix system (Merck Millipore Burlington, MA, USA). The following multiplex kits were used according to the manufacturer’s instructions: Milliplex MAP Akt/mTOR phosphoprotein magnetic bead 11-plex kit, Milliplex MAPK/SAPK signaling 10-Plex kit and Milliplex map β-Tubulin total magnetic beads (all Merck Millipore Burlington, MA, USA). Briefly, the cell pellet was lysed with a cell lysis buffer (Merck Millipore Burlington, MA, USA). The lysate protein concentration was determined with the BCA (bicinchoninic acid) method (Thermo Fischer Scientific, Waltham, MA, USA). A total of 15 µg was used for a multiplex analysis. For acquiring data, the xPONENT 4.2 software (Merck Millipore Burlington, MA, USA) was used.

The following 48 analytes were measured at a protein level by multiplex xMAP technology using the MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel A kit (HCYTA-60K-PX48, Merck, Darmstadt, Germany): sCD40L, EGF, Eotaxin-1/CCL11, FGF-2/FGF-basic, Flt-3 ligand, Fractalkine/CX3CL1, G-CSF, GM-CSF, GROα/CXCL1, IFNα2, IFNγ, IL-1α, IL-1β, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17A/CTLA8, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10/CXCL10, MCP-1/CCL2, MCP-3/CCL7, M-CSF, MDC/CCL22, MIG/CXCL9, MIP-1α/CCL3, MIP-1β/CCL4, PDGF-AA, PDGF-AB/BB, RANTES/CCL5, TGFα, TNFα, TNFβ, and VEGF-A. Multiplex-based assay read-out was performed using the MAGPIX system (Merck) with the xPONENT 4.2 software (Merck) in accordance with the manufacturer’s instruction with overnight incubation of the samples with primary antibodies. A final analysis was carried out with the MILLIPLEX Analyst v5.1 software (Merck). Measurements were performed twice for each sample. Release of the analytes in control and experimental samples was compared with an unpaired two-sample t-test using GraphPad Prism v.8.0.1 (GraphPad Software, Inc., San Diego, CA, USA). The p values ≤ 0.05 were considered significant.