Rtgf β1

Manufactured by BioLegend

Sourced in United States

RTGF-β1 is a recombinant human Transforming Growth Factor beta 1 protein. Transforming Growth Factor beta 1 is a multifunctional cytokine that regulates cell growth, cell differentiation, and other cellular functions.

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5 protocols using rtgf β1

Isolation and Activation of Human T Cells

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Around 3 ml peripheral venous blood was taken from healthy adult donors in accordance with University of Edinburgh ethical agreements (AMREC 20-HV-069). T cells were isolated immediately using the EasySep direct human T cell isolation kit (StemCell Technologies #19661) according to manufacturer’s instructions. Cells were plated in a 96-well round-bottom plate (2 x 10⁵ per well) in complete medium (RPMI, 10% foetal calf serum, 10 units/ml penicillin, 10 μg/ml streptomycin, and 2 mM L-glutamine, all supplied by Gibco, Thermo Fisher UK). EasySep Immunocult stimulation cocktail (StemCell Technologies #10971) and 3 ng/ml rTGFβ1 (Biolegend #781802) were added to all wells, with or without 2.5 μM synthetic human cathelicidin (LL-37). Five days later, cells were washed twice and replated with 50 ng/ml rIL-12 (Biolegend #573002) again with or without LL-37. Three days later, IFN-γ production was assessed by flow cytometry.

Smith K.J., Minns D., McHugh B.J., Holloway R.K., O’Connor R., Williams A., Melrose L., McPherson R., Miron V.E., Davidson D.J, & Gwyer Findlay E. (2022). The antimicrobial peptide cathelicidin drives development of experimental autoimmune encephalomyelitis in mice by affecting Th17 differentiation. PLoS Biology, 20(8), e3001554.

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Th1 and Th17 Cell Differentiation

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CD4⁺ T cells were isolated from the lymphoid tissues of 6–8 wk old mice using EasySep kits (StemCell Technologies) according to the manufacturer’s instructions, except that CD25⁺ nTregs and γδ T cells were also depleted during the process. Purified CD4⁺ T cells were cultured in 6-well plates coated with anti-CD3 (5µg/ml, clone 2c11, BioLegend) and anti-CD28 (5µg/ml, clone 37.51, BioLegend) with complete RPMI-1640 medium for 5 days. For Th1 differentiation, the cultures were supplemented with anti-IL-4 (Frederick National Laboratory) (10µg/ml), rIL-12 (R&D Systems) (5ng/ml), and rIL-2 (Frederick National Laboratory) (200units/ml). For Th17 differentiation, the cultures were supplemented with anti-IL-4 (10µg/ml), anti-IFNγ (10µg/ml, purified in house), rTGFβ1 (2ng/ml), rIL-6 (20ng/ml), rIL-1β (10ng/ml), and rIL-23(5ng/ml) (BioLegend). After 5 days of culture, the cells were rested in complete medium containing rIL-7 (10ng/ml, Frederick National Laboratory) for 2 days before purification and cell transfer.

Li C.R., Mueller E.E, & Bradley L.M. (2014). Islet antigen-specific Th17 cells can induce TNFα-dependent autoimmune diabetes. Journal of immunology (Baltimore, Md. : 1950), 192(4), 1425-1432.

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Immunology Study in C57BL/6 Mice

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Mice were obtained from Japan SLC and maintained in specific pathogen-free conditions in our animal facility. All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee at Waseda University (#2017-A007a, 2018-A090, 2019-A041, 2020-A057, 2021-A003, A22-007, and A23-007). We used 7- to 12-wk-old male or female C57BL/6 mice. Mouse rIL-1β, rIL-2, rIL-4, rIL-6, rIL-21, rIL-23, rTGF-β1, rCXCL13, and anti-ICOS (clone C398.4A) monoclonal Abs (mAbs) were obtained from BioLegend. Mouse rIL-12 and Cellstain CFSE (5- or 6-(N-Succinimidyloxycarbonyl)fluorescein 3’,6’-diacetate) were from Fujifilm Wako Pure Chemistry. mAbs to CD28 (clone 37.51), IL-2 (clone JES6-1A12), IL-4 (clone 11B11), IL-2Rα (clone PC-61.5.3), IL-2Rβ (clone TM-β1), and IFN-γ (clone XMG1.2) were from Bio X Cell. CH-223191, 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) were from Cayman Chemical.

Sakamoto R., Takada A., Yamakado S., Tsuge H., Ito E, & Iwata M. (2023). Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro. PLOS ONE, 18(6), e0287746.

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Adoptive Transfer of Diabetic NOD Tregs

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Total spleen cells from diabetic NOD.Thy1.1 mice were injected i.v. into NOD.Scid mice in a dose containing 4×10⁶ CD3⁺ cells. Antibody treatments were initiated immediately prior to cell transfer. Lymphoid tissues and pancreata were analyzed on Day 14. To generate Tregs, CD4⁺ T cells were isolated from the lymphoid tissues of 6–8-week old NOD.Thy1.1 mice using EasySep kits (StemCell Technologies) according to the manufacturer’s instructions, except that biotin-conjugated anti-CD25 antibody (clone PC61, BioLegend, San Diego) was included in a dose of 0.25µg per 10⁶ cells in a volume of 100µl to deplete endogenous Tregs during the separation. Purified CD4⁺ T cells were cultured in 6-well plates coated with anti-CD3 (145-2C11, 5µg/ml) and anti-CD28 (37.51, 5µg/ml) purchased from BioXcell in complete RPMI-1640 medium for 5 days. The cultures were supplemented with 10µg/ml anti-IFN-γ (XMG1.2 or R46A2), 200units/ml rIL-2 (NCI Biological Resource Branch, Frederick), and 10ng/ml rTGF-β1 (BioLegend) as we previously described [8 (link),9 (link)]. These conditions elicit Tregs uniformly express FoxP3. Tregs were transferred into NOD recipient mice by i.v. injection in a dose of 2×10⁶ cells.

Li C.R., Mueller E.E, & Bradley L.M. (2014). Targeting CD44 Augments the Efficacy of Tregs in Autoimmune Diabetes. Immunology letters, 163(2), 199-205.

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TGF-β1 Regulation of FA BM-MSCs

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BM-MSCs from FA patients or donor were plated into sixwell plates and kept in a 5% CO 2 incubator at 37 °C for 24 h. Cells were then induced with 0.1 or 5 ng/mL of recombinant human TGF-β1 protein (rTGF-β1; BioLegend, USA) containing DMF10 medium for 24 h. Uninduced cells maintained in DMF10 medium were included as controls. The effect of rTGF-β1 treatment on PKNOX2, MEIS1, PBX1 and TGF-β1 expression in BM-MSCs from FA patients (n = 5) and donors (n = 5) was determined. Following induction, BM-MSCs were trypsinized in 0.25% trypsin (Invitrogen, UK) containing 1 mM EDTA (Invitrogen) and washed with PBS (Applichem, Germany), followed by RNA isolation and cDNAs synthesis (i.e. 130 ng RNA was used), according to above protocol. Fold change (FC) in gene expression between induced and control cells were calculated by applying a log transformation to 2 -ΔΔCt [32] . The effect of rTGF-β1 induction on PKNOX2 protein level of BM-MSCs from FA patients (n = 3) and donors (n = 3) was determined using western blot analysis, following the above protocol.

Cagnan I., Cosgun E., Konu O., Uckan D, & Gunel-Ozcan A. (2019). PKNOX2 expression and regulation in the bone marrow mesenchymal stem cells of Fanconi anemia patients and healthy donors. Molecular biology reports, 46(1).

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