ILC2 cells were isolated from the lungs of naïve Foxp3YFPCre mice. These cells were incubated with either IL-33 (10 μg/ml), recombinant GDF15 (R&D) (10 μg/ml) or both. 48 hours later, the expression of IL-13 in ILC2 cells was measured by flow cytometry.
Recombinant gdf15
Manufactured by R&D Systems
Sourced in United States
Recombinant GDF15 is a laboratory research protein produced by R&D Systems. It is a member of the transforming growth factor-beta (TGF-β) superfamily. GDF15 is involved in the regulation of various cellular processes.
Automatically generated - may contain errors
6 protocols using recombinant gdf15
1
Regulation of ILC2 Cell IL-13
2
Isolation and treatment of hepatic stellate cells
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As described previously5 (link),23 (link), hepatic stellate cells (HSCs) were isolated by in situ collagenase perfusion of the liver. After anesthesia by intraperitoneal injection with 100–200 mg/kg ketamine/xylazine cocktails, the liver was perfused in situ with collagenase type I through the portal vein. Non-parenchymal cells were separated by centrifugation at 500 rpm and 4 °C for 5 minutes. Then, the HSCs and Kupffer cells were isolated by differential centrifugation on a density gradient of 11.5% and 20% OptiPrep (Sigma-Aldrich, MO, USA) at 3,000 rpm and 4 °C for 17 minutes. HSCs and Kupffer cells were located in the upper and lower layers, respectively. Kupffer cells were positively purified by labeling with anti-mouse F4/80 microbeads (Miltenyi Biotec) and subjected to real-time PCR analysis. HSCs and Kupffer cells were treated with alcohol (50–200 mM), lipopolysaccharide (10 ng/mL; Sigma-Aldrich, MO, USA), or recombinant GDF15 (100 ng/mL; R&D Systems, Minneapolis, MN, USA).
3
Targeted Knockdown of BMP Signaling Regulators
4
Regulation of ILC2 Cell IL-13
5
Isolation and Treatment of Primary Murine Hepatocytes
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Mouse primary hepatocytes were prepared from 8–10-week-old male WT and GDF15 KO mice (all on a C57BL/6 background) using in situ collagenase perfusion25 (link). Isolated hepatocytes were maintained in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator containing 5% CO2. Primary murine hepatocytes were treated with alcohol (50–100 mM), recombinant GDF15 (100 ng/mL; R&D Systems, Minneapolis, MN, USA) or the oxidative phosphorylation (OXPHOS) complex inhibitors oligomycin (2 µg/mL; Sigma-Aldrich, MO, USA) and rotenone (1 µM; Sigma-Aldrich, MO, USA) for 24 hours.
6
Targeted Knockdown of BMP Signaling Regulators
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All reagents were obtained from GE Dharmacon (Lafayette, CO, USA). ON-TARGETplus human SMARTpools were used for siBMP4 (652; cat#: L-011221-00), siBMP6 (654; cat#; L-021475-00), siBMP7 (655; cat#: L-011592-00), siSTAT3(6774; cat #: L-003544-00), siGDF-15 (9518; cat#: L-019875-00) (referred to as KD#1) and siNTC (cat#: D-001810-10-05) were used for knockdown experiments. ON-TARGETplus individual siRNA human siGDF-15 was used for GDF-15 KD #2 (cat:J-019875-05). siGENOME Control siRNA was used for GAPDH (cat#: D-001140-01-05). Transfections were performed according to the manufacturer’s recommendations using Dharmafect #1 (cat: T-2001) transfection reagent. For monolayer cells, knock-down was performed for 24, 48 or 72 hours before harvesting. For spheroids, knock-down was performed in monolayer culture for 24 hours before trypsinization of cells and subsequent spheroid plating. Spheroids were harvested after 3 days, 4 days after initial siRNA transfection. For GDF-15 rescue experiments, recombinant GDF-15 (R&D systems) was added at time of spheroid plating (24 hours after GDF-15 KD). Spheroids were harvested after 3 days (4 days after initial siRNA transfection). KD efficiencies were confirmed at time of harvest by RT-qPCR and/or by ELISA methods described.
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