50 μL of 2% hydrogel was coated on 96 well plate and 150 μL of various concentration of CCL2 diluted in HBSS (10, 5, 1, 0.5, and 0.1 ng mL−1) was added each well (n = 3). Samples were incubated at 37 °C. At 1, 4, 8, and 24 hour timepoint, 10 μL of sample was aliquoted from each well and kept in −80 °C up to the time of ELISA. Human MCP-1/CCL2 ELISA Kit, (catalog no. RAB0054-1KT, Sigma-Aldrich) was used and manufacturer’s protocol was followed (Fig. 3C and Fig. S4 , ESI † ).
Human mcp 1 ccl2 elisa kit
Manufactured by Merck Group
Sourced in United States
The Human MCP-1/CCL2 ELISA Kit is a laboratory equipment used to quantitatively measure the concentration of the human monocyte chemoattractant protein-1 (MCP-1/CCL2) in biological samples. It is an enzyme-linked immunosorbent assay (ELISA) kit designed to detect and quantify the target analyte.
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3 protocols using human mcp 1 ccl2 elisa kit
1
Quantifying CCL2 Release from Hydrogel
2
Quantification of Inflammatory Cytokines and Mediators
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The CD317+ or CD317− MSCs were plated onto 12-well plates (20 × 104 cells per well), and the cell culture supernatant was collected three days later. The protein levels of CCL2 and TSG6 were measured with a Human MCP-1/CCL2 ELISA Kit (Sigma) and Human TSG6 ELISA Kit (Thermo Fisher Scientific) according to the instructions.
Peripheral blood was collected from the eyes of the mice, and the serum levels of IL-6 (BioLegend), TNF-α (BioLegend), IFN-γ (BioLegend) and IL-1β (BioLegend) were measured with ELISA kits as described previously [31 (link)]. Quantitative PCR (qPCR) was performed as described before after total RNA extraction and reverse transcription [8 (link), 31 (link)]. The primer sequences are listed in Additional file1 : Table S1.
Peripheral blood was collected from the eyes of the mice, and the serum levels of IL-6 (BioLegend), TNF-α (BioLegend), IFN-γ (BioLegend) and IL-1β (BioLegend) were measured with ELISA kits as described previously [31 (link)]. Quantitative PCR (qPCR) was performed as described before after total RNA extraction and reverse transcription [8 (link), 31 (link)]. The primer sequences are listed in Additional file
3
Characterization of Hydrogel-Mediated Cytokine Release
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Supernatant in vitro release profile was characterized by IL-8 and MCP-1 ELISAs. MSU-1.1, HSkMEC.2 and HaCaT cells were seeded in a 24-well plate in 400 μL DMEM—serum-free medium and treated with empty or supernatant-loaded hydrogel spheres while untreated cells and cells treated with a previously optimized dose of 22.5 µg of HATMSC2 supernatant were used as controls. Cells were cultured under hypoxic conditions (1% O2) and, following 24 h, cell culture medium was collected and frozen at −20 °C until ELISA was conducted. The level of supernatant components released from hydrogels was measured using Human IL-8/CXCL8 ELISA Kit and Human MCP-1/CCL2 ELISA Kit (Sigma-Aldrich, Missouri, MO, USA) according to the manufacturer’s protocol. ELISAs were performed using pulled cell culture medium samples from three independent experiments, with two technical repeats.
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