Ntact Gly m 4 allergen (Table 1). Cell cultures had been kept in CO2 -incubator (5 CO2 , 37 C) for 24 h. Culture supernatants from the 96-well plate and basolateral chambers of 24-well plate have been collected 24 h later and stored at -70 C degrees less than one particular week before analytes assessment. Monolayer integrity was checked by measuring TEER prior to and right after the finish of an incubation period.Nutrients 2021, 13,6 ofTable 1. Two stimulation methods, which had been applied to each and every cell culture, except Caco-2 line (only direct stimulation simply because Caco-2 cells had been on the inserts). Stimulation Way Direct stimulation (into 96-well plate) Transepithelial stimulation (into 24-well plate with Caco-2 inserts) Control Gly m four Alone five five Que-3,4 –di-Glc Alone two.5 five Gly m 4 + Que-3,four -di-Glc 5 + 2.5 five + 5 Gly m four Digest five 5- -2.ten. Assessment of Absolute Levels of Cytokines, Chemokines, and Development Components in Cell Cultures Absolute levels of your following 48 analytes were measured by multiplex xMAP technologies working with the MILLIPLEX MAP Cytokine/Chemokine/Growth Issue Panel A kit (IFN-alpha 4 Proteins supplier HCYTA-60K-PXBK48, Merck, Darmstadt, Germany): sCD40L, EGF, Eotaxin-1/CCL11, FGF-2/FGF-basic, Flt-3 ligand, Fractalkine/CX3CL1, G-CSF, GM-CSF, GRO, IFN2, IFN, IL-1, IL-1, 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-1/CCL3, MIP-1/CCL4, PDGF-AA, PDGF-AB/BB, RANTES/CCL5, TGF, TNF, TNF, and VEGF-A. Multiplex-based assay was carried out applying MAGPIX method (Merck, Darmstadt, Germany) using the xPONENT four.two computer software (Merck) in accordance with all the manufacturer’s instruction. Final analysis was performed with the MILLIPLEX Analyst v5.1 application (Merck). Measurements had been performed twice for every sample. two.11. Statistics Absolute values in the analytes in cell culture supernatants had been normalized utilizing a logarithmic transformation by LN function [20] in Microsoft Excel. LN-transformed values had been utilised for comparing the analyte levels in handle and experimental samples by unpaired two-sample t-test making use of Statistica v.10.0.1011.0 analytic package (