PBMC (1 × 106/ml) were cultured in RPMI 1640 supplemented with 10% human serum, 2 mM l -glutamine, and 1% penicillin (Invitrogen Ltd, Paisley, UK) and incubated at 37 °C in a humidified 5% CO2 atmosphere for 2 h in a 12-well plate for monocyte adhesion. After 2 h, non-adhering PBMC were harvested and discarded and monocytes grown on plate were either culture in medium alone (unstimulated) or were or primed with 2 μg/ml lipopolysaccharide (LPS) for 2 h (Sigma-Aldrich, St. Louis, MO, USA) before stimulation with 10 μg/ml of 1-42 amyloid-beta peptide (Aβ42) (Sigma-Aldrich) in the absence/presence of 10 ng/ml of Human Recombinant IL-33 (Biolegend, San Diego, CA, USA) for 24 h at 37 °C in a humidified 5% CO2 atmosphere. After 24 h, supernatants were collected and stored at − 20 °C; adhering cells (monocytes) were collected and prepared for FlowSight analysis.
Recombinant human il 33
Manufactured by BioLegend
Sourced in United States
Recombinant human IL-33 is a cytokine that belongs to the interleukin-1 family. It is produced in Escherichia coli and purified using standard chromatographic techniques.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharide lps, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Alt kit, by Thermo Fisher Scientific (1 mentions)
Recombinant human tgf β1, by BioLegend (1 mentions)
Mouse il 13 elisa kit, by Thermo Fisher Scientific (1 mentions)
L lactic acid, by Merck Group (1 mentions)
Vegf elisa kit, by Thermo Fisher Scientific (1 mentions)
Mouse il 6, by BioLegend (1 mentions)
Sodium l lactate, by Merck Group (1 mentions)
Mcp 1 ccl 2 elisa kits, by BioLegend (1 mentions)
Mcp 1 elisa kits, by BD (1 mentions)
Human il 6, by BD (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by GenScript (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nessy 1, by Abcam (1 mentions)
One hour western detection kit, by GenScript (1 mentions)
5 protocols using recombinant human il 33
1
Amyloid-beta Modulation of Monocyte Responses
2
Human ILC2 Adoptive Transfer for IRI
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Human ILC2s isolated from donor peripheral blood mononuclear cells were cultured in RPMI 1640 medium containing 100 U/ml penicillin–streptomycin, supplemented with 10% human AB serum, plus IL-2 (20 ng/ml), IL-7 (20 ng/ml), and IL-33 (50 ng/ml) for 14 days. Cell-free supernatants were assessed for IL-5 and IL-13 production by ELISA (R&D Systems, Minneapolis, MN, USA). For ILC2 treatment, 5 × 106 human ILC2s were transferred into NSG mice by a single tail-vein injection 1 day before hepatic ischaemia. In parallel, 0.5 × 106 human ILC2s were transfused into NSG mice 5 days before IRI surgery, and then human recombinant IL-33 (0.4 μg/mouse; BioLegend) was administered i.p. daily for 5 consecutive days. NSG mice were humanely culled at Day 1 after IRI surgery. Serum ALT levels were measured using an ALT kit (Thermo Fisher) according to the manufacturer’s instructions.
3
Sensitization and Stimulation of Mast Cells
4
Mouse Cytokine ELISA Assays
5
Quantification and Visualization of IL-33 Isoforms
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A Qubit Protein Assay Kit was used for protein extraction; total protein amount was measured using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). Equal amounts of protein (15 μg) from each sample, or 2 ng of artificial truncated IL-33 (amino acids 112–270, 20 kDa) (Recombinant Human IL33 carrier-free, Biolegend, San Diego, CA, USA) used as positive control to confirm that the bands correspond to IL-33 full length (amino acids 1–270, 34–30 kDa), to the cleaved inactive forms (amino acids 1–178, 22–20 kDa; amino acids 179–270, 13–12 kDa) [2 (link), 3 (link)] or, finally, to the cleaved active forms (amino acids 95–270, amino acids 99–270, and amino acids 109–270; 19–15 kDa) [4 (link)], were separated by 10% SDS-PAGE and transferred to PVDF membranes (Genscript). A pre-stained marker, broad range 11–190 KDa (Cell signaling, Danvers, MA, USA), was also used, PVDF membrane were treated by ONE-HOUR Western detection kit (Genscript) and proteins were visualized using a Chromosensor TMB substrate (Genscript). After protein transfer, PVDF membranes were incubated with a pretreatment solution for 5 min RT and then with the WB solution and an α-human IL-33 Ab (Nessy-1) (Abcam, Cambridge, UK) for 40 min. After three washes, membranes were developed with a TMB substrate.
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