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.
Bio plex pro human cytokine group 2 21 plex panel
Manufactured by Bio-Rad
Sourced in United States
The Bio-Plex Pro Human Cytokine Group II 21-Plex Panel is a multiplex assay designed to simultaneously measure the concentration of 21 different cytokines in human biological samples. The panel provides a comprehensive analysis of various immune and inflammatory markers in a single experiment.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex protein assay system, by Bio-Rad (2 mentions)
Bio plex pro human cytokine 27 plex panel, by Bio-Rad (1 mentions)
Bio plex multiplex reader system, by Bio-Rad (1 mentions)
Bio plex manager software, by Bio-Rad (1 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (1 mentions)
4 protocols using bio plex pro human cytokine group 2 21 plex panel
1
Cytokine Secretion Analysis of Phagocytized Microcapsules
2
Multiplex Cytokine Profiling in Serum
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For the discovery set, the levels of serum cytokines and chemokines were quantified using a Bio-Plex multiplex Reader system (Bio-rad, Milan, Italy) equipped with a Bio-Plex Manager software (BioRad) according to the manufacturer’s instructions. Measurements were performed using two panels (BioPlex Pro Human Cytokine 27-plex panel, Bio-rad and BioPlex Pro Human Cytokine Group II 21-plex panel, Bio-rad) consisting of 48 cytokines and chemokines. All samples were run in triplicate and analyzed according to the manufacturer’s instructions. Serum samples from different time points for the same patient were included on the same assay plate. All of the assays were performed blinded to both the clinicopathologic and prognostic data.
Based on the initial results, serum CCL27 was selected for evaluation in the validation set. For further validation, the human CTACK/CCL27 DuoSet ELISA kit (R&D system, #DY376) was used to determine the concentration of CCL27. Each sample was run in triplicate, and changes from the baseline level to the subsequent individual timepoints were calculated for each cytokine.
Based on the initial results, serum CCL27 was selected for evaluation in the validation set. For further validation, the human CTACK/CCL27 DuoSet ELISA kit (R&D system, #DY376) was used to determine the concentration of CCL27. Each sample was run in triplicate, and changes from the baseline level to the subsequent individual timepoints were calculated for each cytokine.
3
Cytokine and Chemokine Profiling of hAMSCs
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Supernatants from 14-day cell cultures were centrifuged for 10 min at 500 ×g. Chemokines, growth factors, and pro- and anti-inflammatory cytokines (LIF, SCF, SDF-1a, SCGF-b, M-CSF, MCP-3, MIF, MIG, TRAIL, GRO-a; IL-1a, IL-2ra, IL-3, IL-12 (p40), IL-16, IL-18, HGF, TNF-b, b-NGF, IFN-a2, and CTACK) were quantified by fluorescence flow fluorimetry using an automated Bio-Plex Protein Assay System analyzer (Bio-Rad, Hercules, CA, USA) and a commercial test system (Bio-Plex Pro Human cytokine Group II 21-Plex Panel, Bio-Rad, Hercules, CA, USA) in accordance with the manufacturer’s protocol. This cytokine/chemokine profile was previously estimated as niche signal molecules while osteoblastic differentiation of hAMSCs induced by microarc CaP-coated Ti substrates occurred in vitro [5 (link)].
4
Quantifying Viable hAMSCs with Microcapsules
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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.
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.
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