Htgf β, by Thermo Fisher Scientific - Product details

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T Cell Lineage Specification Protocols

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For polarizations using plate-bound αCD3 + αCD28, tissue culture plates were coated with 5 µg/ml αCD3 (145-2C11; BioLegend) and 5 µg/ml αCD28 (37.51; Tonbo), incubated at 37°C for 4 h, and washed three times immediately before use. For differentiation to Th17 cells, naive CD4⁺CD62L^highCD44^low T cells were incubated with IL-6 (20 ng/ml; Peprotech), hTGF-β (5 ng/ml; Peprotech), α IL-4 (10 µg/ml; 11B11; BioLegend), and αIFN-γ (10 µg/ml; XMG1.2; BioLegend), with or without IL-1β (2–10 ng/ml; Peprotech). For differentiation to Th1 cells, naive CD4⁺CD62L^highCD44^low T cells were incubated with IL-12 (10 ng/ml; Peprotech), IL-2 (50 U/ml; eBioscience), and α IL-4 (10 µg/ml; 11B11; BioLegend), with or without IL-18 (0.3–3 ng/ml; Gibco). Th2 cell differentiation was achieved by incubating naive CD4⁺CD62L^highCD44^low T cells with IL-4 (4 ng/ml; Peprotech), IL-2 (50 U/ml; eBioscience), and αIFN-γ (10 µg/ml; XMG1.2; BioLegend).

Deason K., Troutman T.D., Jain A., Challa D.K., Mandraju R., Brewer T., Ward E.S, & Pasare C. (2018). BCAP links IL-1R to the PI3K–mTOR pathway and regulates pathogenic Th17 cell differentiation. The Journal of Experimental Medicine, 215(9), 2413-2428.

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Polarization of Naive CD4+ T Cells

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Cell culture treated plates were coated with αCD3 (5μg mL⁻¹) and αCD28 (5μg mL⁻¹) for 3–4 h at 37°C. CD4⁺CD62L^hi naïve CD4⁺ T cells were isolated from splenocytes using a Mojosort naïve CD4 T cell isolation kit according to manufacturer’s protocol. Purified naïve T cells were plated in antibody-coated plates with appropriate polarizing conditions for 5 days. T cell were cultured in complete RPMI supplemented with 10% FCS. Cytokine cocktails for in vitro polarization: Th1 – IL-12 (10ng mL⁻¹, peprotech), IL-2 (50U mL⁻¹, peprotech), αIL-4 (10μg mL⁻¹, Biolegend); Th17 – IL-6 (20ng mL⁻¹, peprotech), hTGFβ (5ng mL⁻¹, peprotech), αIL-4 (10μg mL⁻¹, Biolegend), αIFNγ (10μg mL⁻¹, Biolegend), IL-23 (20ng mL⁻¹, Biolegend) and IL-1β (20ng mL⁻¹, peprotech), Th2- IL-4 (4ng mL⁻¹, peprotech), IL-2 (50U mL⁻¹, Biolegend), and αIFNγ (10μg mL⁻¹, Biolegend), Th0 – IL-2 (50U mL⁻¹). For co-culture experiments T cells were rested in 10% RPMI supplemented with IL-2 (10U mL⁻¹) for 36hrs before co-culture.

Jain A., Irizarry-Caro R.A., McDaniel M.M., Chawla A.S., Carroll K.R., Overcast G.R., Philip N.H., Oberst A., Chervonsky A.V., Katz J.D, & Pasare C. (2019). T cells instruct myeloid cells to produce inflammasome-independent IL-1β and cause autoimmunity. Nature immunology, 21(1), 65-74.

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Polarization of Murine T Helper Cells

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For in vitro T cell assays, naïve (CD4⁺ CD25⁻ CD44^Lo CD62L^Hi) T cells were puriﬁed by bead enrichment and/or flow cytometric sorting from spleens and lymph nodes from the indicated strains of mice prior to stimulation with plate bound anti-CD3 (2.5 µg/ml; UCSF Monoclonal Antibody Core) and anti-CD28 (2 µg/ml; UCSF Monoclonal Antibody Core). In addition, mIL-12 (20ng/ml, Peprotech) and anti-IL-4 (10µg/ml, 11B11:Tonbo) were used for T_H1 polarization, while hTGFβ (5ng/ml, Peprotech), mIL-6 (20ng/ml, Peprotech), anti-IL-4 (10µg/ml) and anti-IFNγ (10µg/ml, XMG1.2, UCSF hybridoma core) were used for T_H17 polarization. In selected experiments, T cells were labeled with 3 mM CFSE (Invitrogen). siRNA mediated reduction of RIPK3 in Jurkat I9.2 cells (ATCC) was performed using one pulse of 2200 V and 20 ms, using Neon Transfection System (Invitrogen) according to manufacturer’s instructions.

Onizawa M., Oshima S., Schulze-Topphoff U., Oses-Prieto J.A., Lu T., Tavares R., Prodhomme T., Duong B., Whang M.I., Advincula R., Agelidis A., Barrera J., Wu H., Burlingame A., Malynn B.A., Zamvil S.S, & Ma A. (2015). The ubiquitin-modifying enzyme A20 restricts the ubiquitination of RIPK3 and protects cells from necroptosis. Nature immunology, 16(6), 618-627.

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Murine CD4+ T Cell Treg Induction

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CD4+ T cells were isolated from C57/BL6 mouse lymph nodes and spleen using mouse CD3+ T cell enrichment columns (R&D Systems, MN, USA) and CD4+MicroBeads (Miltenyi Biotec) according to the manufacturer’s instructions. Isolated CD4+ T cells were cultured with anti-CD3 (10 µg/mL), anti-CD28 (10 µg/mL) antibodies (Biogems, USA), mIL-2 (5 µg/mL), and hTGF-β (5 µg/mL) (PeproTech, NJ, USA) for 4 days to induce Tregs. On day 4, the Tregs were collected and cultured on the collagen I-coated plate for 18 h. The cells were harvested and stored at −80 °C until use.

Choi J.H., Lee B.S., Jang J.Y., Lee Y.S., Kim H.J., Roh J., Shin Y.S., Woo H.G, & Kim C.H. (2023). Single-cell transcriptome profiling of the stepwise progression of head and neck cancer. Nature Communications, 14, 1055.

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Polarization of Murine T Helper Cells

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For in vitro T cell assays, naïve (CD4⁺ CD25⁻ CD44^Lo CD62L^Hi) T cells were puriﬁed by bead enrichment and/or flow cytometric sorting from spleens and lymph nodes from the indicated strains of mice prior to stimulation with plate bound anti-CD3 (2.5 µg/ml; UCSF Monoclonal Antibody Core) and anti-CD28 (2 µg/ml; UCSF Monoclonal Antibody Core). In addition, mIL-12 (20ng/ml, Peprotech) and anti-IL-4 (10µg/ml, 11B11:Tonbo) were used for T_H1 polarization, while hTGFβ (5ng/ml, Peprotech), mIL-6 (20ng/ml, Peprotech), anti-IL-4 (10µg/ml) and anti-IFNγ (10µg/ml, XMG1.2, UCSF hybridoma core) were used for T_H17 polarization. In selected experiments, T cells were labeled with 3 mM CFSE (Invitrogen). siRNA mediated reduction of RIPK3 in Jurkat I9.2 cells (ATCC) was performed using one pulse of 2200 V and 20 ms, using Neon Transfection System (Invitrogen) according to manufacturer’s instructions.

Onizawa M., Oshima S., Schulze-Topphoff U., Oses-Prieto J.A., Lu T., Tavares R., Prodhomme T., Duong B., Whang M.I., Advincula R., Agelidis A., Barrera J., Wu H., Burlingame A., Malynn B.A., Zamvil S.S, & Ma A. (2015). The ubiquitin-modifying enzyme A20 restricts the ubiquitination of RIPK3 and protects cells from necroptosis. Nature immunology, 16(6), 618-627.

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T Cell Differentiation Protocols

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Naïve CD4 T cells were isolated from mouse spleens and lymph nodes by magnetic bead separation (Miltenyi Biotec) following the manufacturers’ protocols. Cells were cultured in glucose-free RPMI (Gibco) supplemented with 10% heat-inactivated FBS (Corning), 1% penicillin/streptomycin (Gibco), 50μM 2-Mercaptoethanol (Gibco), and either 10mM glucose (Sigma) or 10mM galactose (Sigma). Naïve CD4 T cells were activated with plate-bound anti-CD3e (5μg/ml) and soluble anti-CD28 (2μg/ml) in the presence of mIL-1β (10 ng/ml; R&D Systems), mIL-23 (10ng/ml; R&D Systems), mIL-6 (50ng/ml; R&D Systems), and h TGF-β (5ng/ml; Peprotech) for T_H17 cell polarization; mIL-12 (10ng/ml; R&D Systems) for T_H1 cell polarization, or TGF-β (10ng/ml; Peprotech) and mIL-2 (10ng/ml; R&D Systems) for T_reg polarization. All cells were cultured at 37°C and 5% CO₂. T_H17 differentiation was verified on day 3 or 4, and cytokine expression on day 5, at which point cells were used for subsequent assays.

Hong H.S., Mbah N.E., Shan M., Loesel K., Lin L., Sajjakulnukit P., Correa L.O., Andren A., Lin J., Hayashi A., Magnuson B., Chen J., Li Z., Xie Y., Zhang L., Goldstein D.R., Carty S.A., Lei Y.L., Opipari A.W., Argüello R.J., Kryczek I., Kamada N., Zou W., Franchi L, & Lyssiotis C.A. (2022). OXPHOS Promotes Apoptotic Resistance and Cellular Persistence in TH17 cells in the Periphery and Tumor Microenvironment. Science immunology, 7(77), eabm8182.

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Polarization of Naive CD4+ T Cells

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Cell culture treated plates were coated with αCD3 (5μg mL⁻¹) and αCD28 (5μg mL⁻¹) for 3–4 h at 37°C. CD4⁺CD62L^hi naïve CD4⁺ T cells were isolated from splenocytes using a Mojosort naïve CD4 T cell isolation kit according to manufacturer’s protocol. Purified naïve T cells were plated in antibody-coated plates with appropriate polarizing conditions for 5 days. T cell were cultured in complete RPMI supplemented with 10% FCS. Cytokine cocktails for in vitro polarization: Th1 – IL-12 (10ng mL⁻¹, peprotech), IL-2 (50U mL⁻¹, peprotech), αIL-4 (10μg mL⁻¹, Biolegend); Th17 – IL-6 (20ng mL⁻¹, peprotech), hTGFβ (5ng mL⁻¹, peprotech), αIL-4 (10μg mL⁻¹, Biolegend), αIFNγ (10μg mL⁻¹, Biolegend), IL-23 (20ng mL⁻¹, Biolegend) and IL-1β (20ng mL⁻¹, peprotech), Th2- IL-4 (4ng mL⁻¹, peprotech), IL-2 (50U mL⁻¹, Biolegend), and αIFNγ (10μg mL⁻¹, Biolegend), Th0 – IL-2 (50U mL⁻¹). For co-culture experiments T cells were rested in 10% RPMI supplemented with IL-2 (10U mL⁻¹) for 36hrs before co-culture.

Jain A., Irizarry-Caro R.A., McDaniel M.M., Chawla A.S., Carroll K.R., Overcast G.R., Philip N.H., Oberst A., Chervonsky A.V., Katz J.D, & Pasare C. (2019). T cells instruct myeloid cells to produce inflammasome-independent IL-1β and cause autoimmunity. Nature immunology, 21(1), 65-74.

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Th17 Cell Differentiation and Macrophage Crosstalk

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Naive CD4⁺ T cells isolated by either Miltenyi Biotec or Stemcell Technologies EasyStep Naive CD4⁺ T cell isolation kit (62 (link)). A total of 1 × 10⁵ T cells were plated per well in a 96-well plate and cocultured with Dynabeads Mouse T-Activator CD3/CD28 (Gibco). Naive CD4⁺ T cells were in vitro differentiated into Th17 cells in the presence of anti-CD3 (2 μg/mL, Bio X Cell), anti-CD28 (2 μg/mL, Bio X Cell), hTGF-β (2 ng/mL, Peprotech), and IL6 (20 ng/mL, Peprotech). For macrophage/T cell crosstalk assay, after 1 day of Th17 cell differentiation, T cells were subsequently treated with 50 μL of BMDM culture medium in the conditions of M0 (PBS), M1 (LPS + IFN-γ), M2 (IL4+IL13), M1+FexD (20 μM), M1+OCA (10 μM), M2+FexD (20 μM), and M2+OCA (10 μM) for an additional 2 days. IL1β (10 ng/mL) and IL23 (10 ng/mL) were further added as the Th17 cell differentiation positive control. Cells were then incubated with PMA (50 ng/mL, MilliporeSigma) and ionomycin (500 ng/mL, MilliporeSigma) for 1 hour prior to the addition of GolgiPlug (10 μg/mL, BD Biosciences) for an additional 3–4 hours. Cells were then harvested for flow cytometry and qRT-PCR. Cell culture supernatant was collected for ELISA analysis (see Cytokine and cancer tumor marker measurement).

Dong X., Qi M., Cai C., Zhu Y., Li Y., Coulter S., Sun F., Liddle C., Uboha N.V., Halberg R., Xu W., Marker P, & Fu T. (2024). Farnesoid X receptor mediates macrophage-intrinsic responses to suppress colitis-induced colon cancer progression. JCI Insight, 9(2), e170428.

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Standardized TRAIL and Cytokine Protocols

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For in vitro experiments Recombinant Super Killer Trail (Human: Enzo Lifesciences; Mouse: Adipogene) was dissolved in PBS−/− 1%BSA, kept at −80°C and used 300 ng/ml.
For in vivo experiments Trail Recombinant KillerTRAIL (Alexis) was resuspended in PBS−/− and used 10 mg/kg i.p every 3 days for 2 weeks.
Glucocorticoids (MP Biomedicals) were used 100uM; hIL-10, hIL-1, hIL-2, hIL-4, hIL-6, hTNFα, hINFγ, hTGFβ (Peprotech) 25 ng/ml; LPS (Alexis) 100 ng/ml; Pam3Cys (Vinci Biochem) 2 ug/ml according to the manufacture's instructions.
Apigenin, Quercitin (Indena) and trabectedin (Pharmamar) were resuspended in DMSO and used 40uM, 400uM and 10 nM respectively. Palmitate (SIGMA), Indole-3-Carbinole (I3C) resuspended in DMSO and used 800uM and 100uM respectively.

Liguori M., Buracchi C., Pasqualini F., Bergomas F., Pesce S., Sironi M., Grizzi F., Mantovani A., Belgiovine C, & Allavena P. (2016). Functional TRAIL receptors in monocytes and tumor-associated macrophages: A possible targeting pathway in the tumor microenvironment. Oncotarget, 7(27), 41662-41676.

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In Vitro Expansion of T Cell Subsets

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FACS-sorted CD4⁺ or CD8⁺ (and CD5^lo or CD5^hi) T_N cells (~1−2 × 10⁴/well) were cultured with Dynabeads™ (Gibco) in 24-well plate (Effendorf) with recombinant hIL-1β (50 ng/ml) (PeproTech), hTGF-β (2 ng/ml) (PeproTech), hIL-23 (50 ng/ml) (PeproTech), anti-hIFN-γ (10 μg/ml) (clone B27), and anti-hIL-4 (10 μg/ml) (clone MP4-25D2) for 10 days. Cells were replaced with fresh culture medium every 2−3 days while in culture, and at each replacement, cells were collected, counted, and re-cultured 2 × 10⁶/well under the same conditions until intracellular cytokine assays were performed.

Lee G.W., Kim Y.J., Lee S.W., Kim H.O., Kim D., Kim J., Kim Y.M., Kang K., Rhee J.H., Chung I.J., Bae W.K., Oh I.J., Yang D.H, & Cho J.H. (2024). Developmental self-reactivity determines pathogenic Tc17 differentiation potential of naive CD8+ T cells in murine models of inflammation. Nature Communications, 15, 2919.

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Htgf β

Lab products found in correlation

14 protocols using htgf β

T Cell Lineage Specification Protocols

Polarization of Naive CD4+ T Cells

Polarization of Murine T Helper Cells

Murine CD4+ T Cell Treg Induction

Polarization of Murine T Helper Cells

T Cell Differentiation Protocols

Polarization of Naive CD4+ T Cells

Th17 Cell Differentiation and Macrophage Crosstalk

Standardized TRAIL and Cytokine Protocols

In Vitro Expansion of T Cell Subsets

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