Bio plex protein assay system

Cell culture media was collected at 24 and 48 h post-phagocytosis of various ratios of microcapsules. Collected supernatants were centrifuged at 500 g for 10 min and analyzed by fluorescence flow cytometry by measuring the spontaneous and microcapsule-induced secretion of the following human cytokines and chemokines: LIF, SCF, SDF-1a, SCGF-β, M-CSF, MCP-3, MIF, MIG, TRAIL, Gro-α; IL-1a, IL-2ra, IL-3, IL-12 (p40), IL-16, IL-18, HGF, TNF-β, β-NGF, IFN-α2, and CTACK. Fluorescence flow cytometry was conducted with monoclonal antibodies according to the manufacturer’s instructions for the cytokine assay system (Bio-Plex Pro Human Cytokine Group II 21-Plex Panel, Bio-Rad, Hercules, CA, United States) using an automated processing system (Bio-Plex Protein Assay System, Bio-Rad, Hercules, CA, United States). The concentration of each cytokine was presented in pg/ml, with n = 3–9 per group.

Serum concentrations of 25 humoral factors including 15 cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, TNF-α, IL-1ra, IL-5, IL-7, IL-9, IL-12, IL-15, IL-17), 6 chemokines (IL-8, eotaxin, IP-10, MCP-1, MIP-1α, MIP-1β), and four growth factors (G-CSF, GM-CSF, VEGF, FGF-basic) were determined using a double-beam laser automatic analyser (Bio-Plex Protein Assay System; Bio-Rad, USA). Serum samples were frozen at − 80 °C immediately after collection and were stored until analysis. All samples were thawed on ice, vortexed, centrifuged at 14,000 × g for 10 min at 4 °C, and diluted with the standard diluent in the kit (Bio-Plex Pro Human Cytokine 27-Plex Assay #M500KCAF0Y; ratio 1:4 by volume). Each sample was measured more than twice. The mean values of measurements were used as the representative values for each subject.

Cell culture supernatants were collected on days 2 and 14 and centrifuged at 500× g for 10 min. FC was performed to measure the spontaneous and CaP coating-induced secretion of the following human cytokines and chemokines: Interleukin (IL)-1β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), eotaxin, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma-induced protein 10 (IP-10; C-X-C motif chemokine 10 (CXCL10)), monocyte chemoattractant protein-1 (MCP-1; chemokine (C-C motif) ligand 2 (CCL2)), macrophage inflammatory protein 1 alpha (MIP-1α; CCL3), MIP-1β (CCL4), regulated upon activation, normal T cell expressed and secreted (RANTES; CCL5), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and vascular endothelial growth factor (VEGF).
FC was conducted with mAbs according to the manufacturer’s instructions for the cytokine assay system (Bio-Plex Pro Human Cytokine 27-Plex Panel, Bio-Rad, Hercules, CA, USA) using an automated processing system (Bio-Plex Protein Assay System, Bio-Rad, Hercules, CA, USA). The concentration of each cytokine is presented in pg/mL.

The content of targeted proteins in cell culture supernatants after 24 h cultivation in serum-free medium was measured by continuous-flow fluorometry with a double-beam laser automatic analyzer Bio-Plex Protein Assay System (Bio-Rad, USA) using commercial kits (Bio-Plex Pro 27-Plex and 21-Plex Assay, Bio-Rad, USA) in accordance with the manufacturer's instructions.

The levels of 13 cytokines—IL-1 (IL-1 $\mathsf{\beta }$ ), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-17A, IFN- $\mathsf{\gamma }$ , TGF- $\mathsf{\beta }$ and TNF- $\mathsf{\alpha }$ —were evaluated both in serum and in culture supernatants using a two-laser automated analyzer (Bio-plex Protein Assay System, Bio-Rad, Hercules, CA, USA) with commercial test systems (determined dynamic range 0.2–3200 pg/mL) in accordance with the manufacturer’s instructions. All reactions were performed in a 96-well plate format. The amount of cytokines in the test samples was determined using standard calibration dilutions, and cytokine concentrations were calculated automatically using the Bio-Plex Manager software.

Quantitative determination of leptin was evaluated by flow fluorimetry on a «Bio-Plex Protein Assay System» (Bio-Rad, USA) automated laser analyzer using Bio-Plex Pro Human Diabetes 10-Plex Assay commercial test system (Bio-Rad, USA). The method consists in the binding of the studied molecules with specific antibodies adsorbed on the surface of microspheres (magnetic granules), which allows to determine up to 100 analytes in one well. The results were read using an automatic Bio-Plex-200 System microplate photometer (Bio-Rad, USA) and the Bio-Plex Manager software (Bio-Rad, USA). Leptin concentrations were determined using a standard curve (determined dynamic range of 2–32,000 pg / ml) in accordance with the instructions of the manufacturer.

The in vitro viability of hAMSCs loaded with different doses of FITC-labeled microcapsules was estimated using a CountessTM Automated Cell Counter (Invitrogen, Carlsbad, CA, USA) after staining with 0.4% trypan blue. The percentage of viable and dead (stained) cells was measured after they were harvested with 0.05% trypsin in 0.53 mM of EDTA and washed twice with PBS.
Supernatants from 24, 48, and 72h MSC cultures loaded with different ratios of microcapsules were collected and centrifuged at 500× g for 10 min. Chemokines GRO-α, MIF, and SDF-1α were determined by fluorescence flow fluorimetry using an automated Bio-Plex Protein Assay System (Bio-Rad, Hercules, CA, USA) and a commercial assay system (Bio-Plex Pro Human cytokine Group II 21-Plex Panel, Bio-Rad, Hercules, CA, USA, for GRO-α, MIF, SDF-1a, LIF, SCF, SCGF-β, CTACK, M-CSF, MCP-3, MIG, TRAIL, IL-1a, IL-2ra, IL-3, IL-12 (p40), IL-16, IL-18, HGF, TNF-β, β-NGF, and IFN-α2), according to the manufacturer’s protocol.