Naïve CD4+ T cells were sorted from the splenocytes of healthy BALB/c mice using a Naïve CD4+ T cell isolation kit (Miltenyi Biotec MACS, Germany). For detection Th17 differentiation and proliferation, cells were cultured with or without 5 μM carboxyfluorescein succinimidyl ester (CFSE) labeling according to the manufacturer’s protocols (Livak and Schmittgen, 2001 (link)). The cultured condition of cells after sorting was IMDM medium comprised of 10% heat inactivated FBS (Gibco, United States), 2 mM of l -glutamine (Sigma), 0.1 mM nonessential amino acids (Sigma), 1% penicillin/streptomycin, and 100 µM β-mercaptoethanol (Sigma-Aldrich). For Th17 cells’ differentiation, anti-mouse IFN-γ (10 μg/ml; Biolegend, United States), anti-mouse IL-4 (10 μg/ml; Biolegend, United States), recombinant mouse IL-6 (60 ng/ml; Biolegend, United States), and recombinant mouse TGF-β1 (5 ng/ml; Biolegend, United States) were added in 96-well plates with plate-bound anti-mouse CD3 (5 μg/ml; Biolegend, United States) and anti-mouse CD28 (5 μg/ml; Biolegend, United States). After 72 h, cells were collected for further experiments.
Recombinant mouse tgf β1
Manufactured by BioLegend
Sourced in United States
Recombinant mouse TGF-β1 is a protein produced in a laboratory setting that is biologically equivalent to the naturally occurring mouse Transforming Growth Factor beta 1 cytokine. It is a multi-functional protein that plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (1 mentions)
Na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Anti mouse ifn γ, by BioLegend (1 mentions)
Recombinant mouse il 6, by BioLegend (1 mentions)
Anti mouse il 4, by BioLegend (1 mentions)
Anti mouse cd3, by BioLegend (1 mentions)
Anti mouse cd28, by BioLegend (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Recombinant murine vegf165, by Thermo Fisher Scientific (1 mentions)
Cellmatrix type 1 a, by Nitta Gelatin (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Recombinant murine pdgf bb, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by MedChemExpress (1 mentions)
5 protocols using recombinant mouse tgf β1
1
Th17 Cell Differentiation and Proliferation
2
3D Culture of Mouse Endothelial Cells
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We used MS1 (MILE SVEN 1, ATCC CRL-2279), an endothelial cell line derived from mouse pancreatic islets, or sorted murine lung cells for cell culture experiments. DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin–streptomycin were added to 40 ng/mL of TGF-β (Recombinant Mouse TGF-β1, BioLegend), 50 ng/mL of PDGF (Recombinant Murine PDGF-BB, PeproTech, Cranbury, NJ, USA), or 50 ng/mL of VEGF (Recombinant Murine VEGF165, PeproTech). For gene silencing using siRNA, we used Silencer Select Pre-Designed siRNA (Nestin or Notch3, Thermo Fisher Scientific) and Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). The method of 3D culture was as follows: the cell suspension was mixed with Cellmatrix type I-A (Nitta Gelatin, Osaka, Japan), seeded onto 12 or 24 well-culture plates, incubated at 37 °C for 30 min, and treated with growth factor-containing media. The culture duration depended on cell types, 7 to 21 days in general. The media were replaced twice or three times per week. Vascular densities were determined using Vessel Analysis (Image J, National Institutes of Health, Bethesda, MD, USA) as described in previous studies [35 (link)–37 (link)].
3
TGFβ-Induced IFNβ Production in Irradiated Cells
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RAW 264.7 cells or BMDMs were cultured as described above and then treated with recombinant mouse TGFβ1 (Biolegend). Fifteen min later, the cells were irradiated with 8 Gy and then treated with MT1 or IgG at 0.1 µg/mL. Treatments with TGFβ and MT1 or IgG were renewed 24 hours after irradiation. Supernatants were collected 1 day later. Supernatants of BMDMs were 5-fold concentrated using the Amicon Ultra-0,5 Membrane Ultracel-10 columns (Merck Millipore) and the IFNβ concentration was evaluated using the VeriKine Mouse Interferon Beta HS ELISA Kit (PBL assay science).
4
Asthma Mouse Model with Allergen Challenges
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Asthma mouse model induced by cockroach (CRE, Greer Laboratories Inc.) and HDM exact (Stallergenes Greer) was established as previously reported (71 (link)). Mice were sacrificed, and samples were collected on the next day after the last allergen challenge. In a separate experiment, mice were treated with 25 ng recombinant mouse TGF-β1 (BioLegend) dissolved in PBS by intratracheal administration or with 20 μg 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB, CAS# 107254-86-4) by i.p. injection 30 minutes prior to allergen challenge. Vehicle-treated mice received PBS.
5
Evaluating SHR-1701 in Lung Cancer
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To verify that SHR‐1701 blocked the TGF‐βR in mouse tumor cells, CMT167 cells were treated with SHR‐1701 (10 μg/mL) or control IgG at 37°C for 40 min. In order to ensure that TGF‐βR was indeed blocked by SHR‐1701 in an original activated state, the cells were incubated with recombinant mouse TGF‐β1 (8 ng/mL; #763104, Biolegend) for 40 min with or without SHR‐1701. Similarly, for the sake of determinizing the activation status of the Akt signaling pathway in tumor cells, CMT167 cells incubated with SHR‐1701 or control IgG for 40 min were transferred to a plate coated with recombinant mouse PD‐L1‐Fc chimera (5 μg/mL; #758206, Biolegend). The proteins were purified after 24 h followed by western blotting analysis.
To verify that SHR‐1701 rescued the anti‐tumor function of CD8+ T cells in lung cancer patients, PBMCs extracted from patients were pretreated with 10 μg/mL SHR‐1701, 10 μg/mL anti‐PD‐1 Abs (#AF1086, R&D systems, Minneapolis, Minnesota, USA) or 1 μmol/L TGF‐βR inhibitors (#S1476, Selleck, Houston, Texas, USA) for 3h at 37℃.Then PBMCs were treated with Phorbol‐12‐myristate‐13‐acetate (PMA, 10 ng/mL; #P1585, Sigma‐Aldrich) and ionomycin (1 μg/mL; #HY‐13434, MedChemExpress, Princeton, NJ, USA) for 4 h before the CD8+ and CD4+ T cell were isolated and collected for further examinations.
To verify that SHR‐1701 rescued the anti‐tumor function of CD8+ T cells in lung cancer patients, PBMCs extracted from patients were pretreated with 10 μg/mL SHR‐1701, 10 μg/mL anti‐PD‐1 Abs (#AF1086, R&D systems, Minneapolis, Minnesota, USA) or 1 μmol/L TGF‐βR inhibitors (#S1476, Selleck, Houston, Texas, USA) for 3h at 37℃.Then PBMCs were treated with Phorbol‐12‐myristate‐13‐acetate (PMA, 10 ng/mL; #P1585, Sigma‐Aldrich) and ionomycin (1 μg/mL; #HY‐13434, MedChemExpress, Princeton, NJ, USA) for 4 h before the CD8+ and CD4+ T cell were isolated and collected for further examinations.
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