Human renal proximal tubular epithelial cells (HK-2 cells, American Type Culture Collection, Manassas, VA, USA) were cultured, as previously described. Briefly, cells were passaged approximately every three to four days in 100-mm dishes containing combined Dulbecco’s modified Eagle’s (DMEM) and Hams F-12 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Life Technologies; Gaithersburg, MD, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich). The cells were then incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 °C for 24 h, and sub-cultured until 70–80% confluence. For treatment with recombinant proteins or chemicals, cells were plated onto 60-mm dishes in a medium containing 10% FBS and incubated for 24 h. The cells were then incubated in DMEM-F12 medium with 2% FBS and treated with rhAngII (1 μg/mL; Bachem, Bubendorf, Switzerlandsource) or rhTGFβ (2 ng/mL; R&D Systems) for an additional 16 h. Olmesartan medoxomil (5 ng/mL) was added 1 h prior to rhTGFβ treatment.
Rhtgf β
Manufactured by R&D Systems
Sourced in United States, United Kingdom
RhTGF-β is a recombinant human protein that serves as a growth factor. It is a member of the transforming growth factor beta (TGF-β) superfamily.
Automatically generated - may contain errors
Lab products found in correlation
Rhil 2, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Anti cd28 clone 37, by Thermo Fisher Scientific (3 mentions)
Multiskan sky, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Hk 2 cell, by American Type Culture Collection (2 mentions)
Cpg odn 2006, by InvivoGen (2 mentions)
Rhapril, by R&D Systems (2 mentions)
Rhbaff, by R&D Systems (2 mentions)
Anti cd3 clone 145 2c11, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Rhil 2, by Roche (2 mentions)
Interleukin 2 (il 2), by Merck Group (2 mentions)
L3t4 microbeads, by Miltenyi Biotec (2 mentions)
Anti cd3 145 2c11, by BD (2 mentions)
Anti cd28 37.51, by BD (2 mentions)
Rhil 15, by R&D Systems (2 mentions)
Rhil 2, by R&D Systems (2 mentions)
Cd4 cd25 regulatory t cell kit, by Miltenyi Biotec (2 mentions)
32 protocols using rhtgf β
1
HK-2 Cell Culture and Stimulation
2
Optimizing CAR T Cell Therapy with TGF-β
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T cells were activated and stimulated for 2 days with CD3/CD28 antibodies and transduced with the specific CAR constructs. On the day of the transduction (day 0), T cells were incubated with human recombinant TGFβ1 (rhTGFβ) (R&D System, Abingdon, UK, 7754-BH) at 1, 2.5, 5, 10, or 15 ng/mL. Media-containing rhTGFβ was refreshed twice a week until the end of the experiment. T cell proliferation was measured every 7 days for 3 weeks using 123 count eBeads™ (Thermo Fisher Scientific, Oslo, Norway, 01-1234-42) and analyzed with flow cytometry according to the manufacturer’s instructions. At day 21, CAR T cells were used in a 48 h killing assay at an E:T ratio of 1:1 with 22Rv1 target cells.
3
Regulatory T Cell Induction Protocol
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CD4+ T cells were purified from the spleens of Foxp3eGFP reporter mice and then were activated with plate-bound anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) antibodies in the presence of rhTGF-β (5 ng/mL; R&D Systems) and rhIL-2(100 IU/mL, NIH/NCI). The cultured CD4+ cells were incubated at 37°C in 5% CO2 for 4–5 days before sorting the GFP+ cell fractions.
4
Transcriptional Profiling of TGF-β-Stimulated Fibroblasts
5
Naïve B Cell Differentiation
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Cells are cultured in IMDM supplemented with 10% FCS, 100 IU/ml penicillin and 50 μg/ml streptomycin. PBMCs derived from healthy donors were isolated using ficoll-Paque. CD19+ B cells were subsequently MACS-isolated using CD19 beads (Miltenyi Biotec), where after CD19+IgD+CD27− naïve B cells were FACS-sorted. 2 ×105 Naïve B cells were cultured for 6 days on 50.000 irradiated CD40L-expressing fibroblasts in the presence of 50 ng/ml recombinant mouse IL-21, 0,5 ng/ml rhTGF-β (R&D Systems), 100 ng/ml rhAPRIL (R&D Systems) and 100 ng/ml rhBAFF (R&D Systems). When stated, blocking antibodies to TACI and BCMA (both 10 μg/ml, R&D systems) were added to the cultures. In some experiments, B cells were additionally stimulated for 2 days with 10 ng/ml LPS (Invivogen), 10 μg/ml CpG (ODN2006, Invivogen) or medium as control after the 6 day culture protocol.
6
Foxp3 Treg Cell Differentiation
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CD4+CD44loFoxp3− naive T cells from Foxp3-Tocky mice were isolated by cell sorting, and 105 cells were cultured on anti-CD3 (clone 1452C11; 2 µg/ml; eBioscience) and anti-CD28 (clone 37.51; 10 µg/ml; eBioscience)–coated 96-well plates (Corning) in the presence of 100 U/ml rhIL-2 (Roche) and 2 ng/ml rhTGFβ (R&D) for 0–48 h in a final volume of 200 µl RPMI 1640 (Sigma-Aldrich) containing 10% FCS and penicillin/streptomycin (Thermo Fisher Scientific).
Mature Foxp3+ Treg cells from Foxp3-Tocky mice were isolated by cell sorting, and 105 cells were cultured on anti-CD3 (clone 145.2C11; 2 µg/ml) and anti-CD28 (clone 37.51; 10 µg/ml) –coated 96-well plates in the presence of 100 U/ml rhIL-2 for 0–48 h in a final volume of 200 µl RPMI 1640 containing 10% FCS and penicillin/streptomycin.
Mature Foxp3+ Treg cells from Foxp3-Tocky mice were isolated by cell sorting, and 105 cells were cultured on anti-CD3 (clone 145.2C11; 2 µg/ml) and anti-CD28 (clone 37.51; 10 µg/ml) –coated 96-well plates in the presence of 100 U/ml rhIL-2 for 0–48 h in a final volume of 200 µl RPMI 1640 containing 10% FCS and penicillin/streptomycin.
7
Generation of Induced Regulatory T Cells
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Induced Tregs (iTregs) were generated as described previously.7 (link) Briefly, CD4+ cells were isolated from B6 spleen and lymph nodes by magnetic bead sorting (L3T4 Microbeads, Miltenyi Biotec), and cultivated in plates coated with 10 µg/ml anti-CD3 (145-2C11), 1 µg/ml anti-CD28 (37.51) (BD Pharmingen) in the presence of 100 U/ml interleukin-2 (IL-2; Sigma) and 5 ng/ml recombinant human transforming growth factor-beta (rhTGF-β; R&D Systems) for 5 days. At the end of culture, the Treg-enriched cell population was used for therapeutic administration without additional sorting steps. FoxP3 expression was usually >80%. Then 3 × 106 cells were injected intravenously per mouse.
8
Monocyte and NK Cell Activation for ADCC
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Purified monocyte subsets and NK cells were pre-incubated at 37 °C for 5 hrs with 100 ng/ml LPS (E. coli 0111:B4; Sigma Aldrich), 10 μg/ml R848 (Invivogen), 100 ng/ml rhIL-12 (Immunotools), 100 ng/ml rhIL-15 (R&D Systems), 20 ng/ml rhIFN-γ (Immunotools), 8 μg/ml S100A9 (Origene Technologies) or 100 ng/ml HMGB1 (R&D Systems) before they were used for ADCC assay with BATDA-labeled GM2-coated A549 as target cells at an E:T ratio of 10:1. Freshly isolated CD16− monocytes were cultured in complete IMDM either in the absence or presence of 50 ng/ml rhM-CSF (Immunotools), 50 ng/ml rhTGF-β (R&D Systems) or 50 ng/ml rhIL-10 (Immunotools) at 37 °C for 24 hrs before their ADCC activity was assessed on trastuzumab-coated SKBR3 at a 10:1 E:T ratio.
9
TGF-β Stimulation of si-Trim59 Cells
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Recombinant human transforming growth factor beta (Rh TGF-β) (R&D Systems, Minneapolis, MN, USA) was reconstituted at 20 µg/mL in sterile 4 mM HCl containing 0.1% bovine serum albumin for storing and use. Cells of the si-Trim59 group cells were seeded in 6-well plates at 40% confluence on the day before stimulation. At 48 h after 2 ng/mL Rh TGF-β stimulation, cells were harvested for transwell assay. Si-Trim59 group cells stimulated with Rh TGF-β were defined as si-Trim59 + TGF-β group.
10
Quantification of TGFβ Secretion by ELISA
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Secretion of total and active TGFβ into conditioned medium was quantified using ELISA assays according to the manufacturer's instructions (BosterBio). rhTGFβ (R&D systems #240-B-002) was used for the standards. For active TGFβ levels, conditioned medium was incubated with 1 N HCl and 1.2 N NaOH/0.5 M HEPES. Samples and standards were added to TGFβ monoclonal antibody pre-coated 96-well plates and incubated for 90 min at 37°C. Biotinylated antibodies were added and incubated at 37°C for 1 hr. After washing with 0.01 M PBS, ABC working solution was added and incubated at 37°C for 30 min. After washing, TMB color developing agent was added and the plate incubated at 37°C in the dark for 30 min. TMB stop solution was added and the OD absorbance at 450 nm was recorded in a microplate reader (Promega).
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