Latent tgf β

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Latent TGF-β is a protein complex that contains the inactive form of Transforming Growth Factor-beta (TGF-β). TGF-β is a multifunctional cytokine involved in various cellular processes. This product can be used to study the regulation and activation of TGF-β signaling.

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TGF-β (636 citations)

3 protocols using latent tgf β

Isolation and Polarization of Murine T Cell Subsets

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Naïve CD4⁺ T cells were isolated and purified from the spleens of C57BL/6 mice using a naïve CD4⁺ T cell isolation kit (Stemcell Technologies, Cat # 19765), and then cultured in 96-well round bottom plates with 0.5 μg/mL plate-bound anti-CD3 (Biolegend, Cat # 100340). T cells were cultured in RPMI medium (ThermoFisher Scientific, Cat #11879020) supplemented with indicated concentrations of glucose and 10% fetal bovine serum. Inducible Treg (iTreg) polarizing cultures also contained 1 μg/mL anti-CD28 (Biolegend, Cat # 102116), and either 15 ng/mL latent TGF-β (R&D Systems, Cat # 299-LT-005) or 0.5 ng/mL active TGF-β (PeproTech, Cat # 100–21). Th1 polarizing cultures contained 1 μg/mL anti-CD28, 5 μg/mL anti-IL-4 (Tonbo Biosciences, Cat # 70–7041), 3 ng/mL IL-12 (PeproTech, Cat # 210–12), and 5 ng/mL IL-2 (PeproTech, Cat # 212–12). Mixed T cell cultures contained 1 μg/mL anti-CD28, 0.5 ng/mL active or 15 ng/mL latent TGF-β, and 3 ng/mL IL-12. As indicated, some cultures included 15 mM lactate (Sigma-Aldrich, Cat # L4263) or 20 μM MCT1/2 inhibitor (Tocris Bioscience, Cat # 4960). Th17 differentiation was carried out with 1 μg/mL anti-CD28, 5 ng/mL active TGF-β, and 50 ng/mL IL-6.

Pitmon E., Meehan E.V., Ahmadi E., Adler A.J, & Wang K. (2023). High glucose promotes regulatory T cell differentiation. PLOS ONE, 18(2), e0280916.

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Induction of Th17 Cells from Naive T Cells

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Splenic DCs were sorted to more than 95% purity using the CD11c Positive Isolation Kit (Miltenyi Biotec). Naive 2D2 T cells were isolated from the lymph nodes and spleens of 2D2 TCR transgenic mice using the CD4⁺CD62L⁺ T-cell isolation kit (Miltenyi Biotec). Co-culture of CD4⁺ T cells with splenic DCs has been described previously (29 (link), 30 (link)). Briefly, purified naive 2D2 CD4⁺ T cells were cultured with splenic DCs at a ratio of 5:1 in the presence of the MOG_35–55 peptide (10 µg/ml), IL-6 (50 ng/ml; Peprotech), IL-1β (10 ng/ml; Peprotech), FICZ (6-formylindolo [3,2-b] carbazole; 300 nM; Biomol) and TGF-β (1 ng/ml; R&D Systems) or latent-TGF-β (50 ng/ml). For RGD blockade experiments, cRGD peptide was added at 4 µg/ml. For the co-culture assay, co-cultured cells were maintained in X-VIVO™ 15 Chemically Defined, Serum-free Hematopoietic Cell Medium (Lonza). The 2D2 T cells were analyzed by flow cytometry after 3 days of culture. Cytokines in the culture supernatants were measured using an IL-17A ELISA kit (R&D, USA). The activation of TGF-β was measured with the Plasminogen Activator Inhibitor-1 Promoter Luciferase Assay (details described below).

Su P., Chen S., Zheng Y.H., Zhou H.Y., Yan C.H., Yu F., Zhang Y.G., He L., Zhang Y., Wang Y., Wu L., Wu X., Yu B., Ma L.Y., Yang Z., Wang J., Zhao G., Zhu J., Wu Z.Y, & Sun B. (2016). Novel function of Extracellular matrix protein 1 in suppressing Th17 cell development in experimental autoimmune encephalomyelitis. Journal of immunology (Baltimore, Md. : 1950), 197(4), 1054-1064.

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Intestinal Epithelial Cell Culture Protocol

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T84 intestinal epithelial cells purchased from the American Type Culture Collection (Manassas, Va) were grown and maintained in a 1:1 (vol:vol) mixture of Dulbecco modified Eagle medium and Ham F-12 medium containing L-glutamine (0.365 g/L) and sodium bicarbonate (2.438 g/L), supplemented with fetal bovine serum (10%) and 2% penicillin-streptomycin at 37°C in 5% CO 2 (all from Invitrogen, Burlington, Ontario, Canada).
Caco-2 bbe (American Type Culture Collection, labeled Caco-2 hereafter) cells were grown and maintained in Dulbecco modified Eagle medium containing L-glutamine (0.584 g/L) and supplemented with 10% fetal bovine serum, 2% penicillin-streptomycin, sodium pyruvate (1 mM), and human transferrin (0.01 mg/mL) at 37°C in 5% CO 2 (all from Invitrogen).
Recombinant human TGF-β1 (TGF-β throughout text, unless otherwise specified), TGF-β2, or latent TGF-β (R&D Systems Inc, Minneapolis, Minn) were added into the basolateral side of wells (0.1 -100 ng/mL, 1 -72 h), and transepithelial electrical resistance (TER) was recorded throughout the experimental period.
Enterohemorrhagic E. coli O157:H7 strain CL56 ( 11 ) was maintained on 5% sheep blood agar plates (Oxiod, Nepean, Ontario, Canada) at 4°C, cultured overnight in nonaerated Luria-Bertani broth at 37°C, and then added to the apical compartment of intestinal epithelia at approximately 1 × 10 8 colony-forming units/monolayer.

Howe K.L., Lorentz R.J., Assa A., Pinnell L.J., Johnson-Henry K.C, & Sherman P.M. (2015). Transforming growth factor-β1 protects against intestinal epithelial barrier dysfunction caused by hypoxia-reoxygenation. Shock (Augusta, Ga.), 43(5).

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