anti-tgfβ1 neutralizing antibody  (R&D Systems)

Journal: International Journal of Biological Sciences

Article Title: Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution

doi: 10.7150/ijbs.69890

Figure Lengend Snippet: TGF and FGF up-regulate PD-L1 and skew macrophage polarization . (a, b) Expression of PD-L1 on the surface of fibroblast-like cells detected by FCM. (c, d, f, g) Expression of CD86 and CD206 in WT-MEF or PD-L1 KO MEF detected by FCM. Macrophages were co-cultured with untreated, or FGF-2 and TGF-β1 pretreated fibroblasts were washed with PBS, blocked in 5% FBS and two color-stained to identify M1 (CD86) and M2 (CD206) macrophage subpopulations.(e, h) Expression of CD16, CD86 and CD206 in macrophages co-cultured with pretreated fibroblast-like cells detected by immunofluorescence staining.(i) Pretreating WT or PD-L1-/- fibroblasts (fixed with Paraformaldehyde or not) affect activated macrophages TNF-α, IL-6 and IL-10 production in a contact-dependent manner. *p < 0.05, **p < 0.01.

Article Snippet: 20 mg/mL anti-FGF-2 neutralizing antibody (Sigma Aldrich, St. Louis, MO, USA) and 50 mg/mL anti-TGFβ1 neutralizing antibody (Thermo Fischer Scientific; former Savant, MA, USA) were incubated along with Wound exudate in FACS, where indicated.

Techniques: Expressing, Cell Culture, Staining, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution

doi: 10.7150/ijbs.69890

Figure Lengend Snippet: TGF-β1 together with FGF-2 significantly upregulated fibroblast-like cells PD-L1 at translational levels. (a, b) Polysome profiles of MEF cells treated 24 h with FGF-2 and TGF-β1. One representative profile from three independent experiments is shown. Percentage of transcripts in each polysomal fraction obtained by sucrose-gradient ultracentrifugation was quantified by qRT-PCR (n = 3). (c) Western blot analysis of the indicated proteins in MEF when stimulated by FGF-2 and TGF-β1. Representative blots from two independent experiments are shown. (d, e) PD-L1 is visualized by flow cytometry in MEF cells stimulated by FGF-2 and TGF-β1 and eIF4E inhibitor. One representative experiment of two is shown. *p < 0.05,**p < 0.01.

Article Snippet: 20 mg/mL anti-FGF-2 neutralizing antibody (Sigma Aldrich, St. Louis, MO, USA) and 50 mg/mL anti-TGFβ1 neutralizing antibody (Thermo Fischer Scientific; former Savant, MA, USA) were incubated along with Wound exudate in FACS, where indicated.

Techniques: Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: Fibroblast-like cells Promote Wound Healing via PD-L1-mediated Inflammation Resolution

doi: 10.7150/ijbs.69890

Figure Lengend Snippet: PD-L1 positive fibroblast-like cells pretreated by TGF-β1 and FGF-2 promote chronic refractory wound healing in vivo. (a) Representative photographs showing macroscopic excisional wound closure treated with PD-L1 positive and negative MEF in PD-L1-/- C57 BL6 mice. The wild-type and PD-L1-/- mice fibroblast-like cells were isolated and cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin and 50 units/mL streptomycin (Sigma). Followed by pretreated with TGF-β1 and FGF-2, approximately 1x 10 5 fibroblast-like cells were further suspended in saline and sprayed over the wound area of the mice. (b) Planimetric analysis of wound photographs reveals significantly delayed at 3-5 days after wounding when PD-L1-/- MEF were used. (c) The schematic diagram of the mechanism of PD-L1 regulating wound healing.

Article Snippet: 20 mg/mL anti-FGF-2 neutralizing antibody (Sigma Aldrich, St. Louis, MO, USA) and 50 mg/mL anti-TGFβ1 neutralizing antibody (Thermo Fischer Scientific; former Savant, MA, USA) were incubated along with Wound exudate in FACS, where indicated.

Techniques: In Vivo, Isolation, Cell Culture, Modification, Saline

Journal: Scientific Reports

Article Title: Toll-like Receptor-4 Activation Boosts the Immunosuppressive Properties of Tumor Cells-derived Exosomes

doi: 10.1038/s41598-019-44949-y

Figure Lengend Snippet: Expression of TGFβ on the surface of exosomes and its effect on regulatory T cells expansion. The expression of TGFβ1 ( A ) and TGFβ2 ( B ) on the surface of exosomes were evaluated using the TGF-β Magnetic Luminex Performance Assay on exosomes released by unstimulated (CTRL, white column) o LPS-activated tumor cells (LPS, black column). Data are shown as mean (n = 8) ± SD. *Difference with exosomes released by unstimulated tumor cells, P < 0.05. ( B ) Exosomes were coupled to ExoFlow beads, stained with monoclonal antibodies against TGFβ1 and analyzed by FACS. Representative plots of the FACS analyses for staining of control (black line) and of LPS-derived exosomes (red line) are shown. ( D–F ) CD4+ T cells, isolated from PBMCs by negative selection, were stimulated with anti-CD3, anti-CD28 and IL-2 in the presence of exosomes released by unstimulated (CTRL, white column) o LPS-activated tumor cells (LPS, black column). The percentage of effector (CD4+/CD25−/FoxP3−) or regulatory T cells (CD4+/CD25+/FoxP3+) was determined by fluorescence activated flow cytometry (FACS) on day 4. Representative plots for CD25 and Foxp3 staining are shown and histograms represent the ratio between the percentage of effector and regulatory T cells (T eff /T reg ). In a separate set of experiments, exosomes were pre-treated with neutralizing anti-TGFβ1 antibody. Data are shown as mean (n = 6) ± SD. *difference with the untreated cells, P < 0.05 ; **difference with untreated cells, P < 0.005 , $ difference with exosomes treated cells released by LPS-activated cells.

Article Snippet: In a separate set of experiments, SW480-, SW620- and U87-MG-derived exosomes were pre-incubated for 15 min at 37 °C with 0.5 µg/ml of anti-TGFβ1 neutralizing antibody (R&D system) before adding to cells.

Techniques: Expressing, Luminex, Staining, Derivative Assay, Isolation, Selection, Fluorescence, Flow Cytometry

Journal: PLoS ONE

Article Title: Role of 4-1BB Receptor in the Control Played by CD8 + T Cells on IFN-γ Production by Mycobacterium tuberculosis Antigen-Specific CD4 + T Cells

doi: 10.1371/journal.pone.0011019

Figure Lengend Snippet: Pooled spleen cells of NP-mice, unfractionated or depleted of CD4 + or CD8 + T cells by using magnetic beads as described in (panels B and C), were re-stimulated with Ag85B protein (5 µg/ml) in the presence of agonistic anti-4-1BB mAb (5 µg/ml) or rat IgG2a control Ab (5 µg/ml). Culture supernatants were harvested at 48 h for IL-2, IL-4, at 72 h for IFN-γ, IL-6, IL-10, MIP-1β, TGF-β1, TNF-α, XCL-1 and at 96 h for IL-17 detection by specific quantitative sandwich ELISA Kits. Data are presented as mean of 3 independent experiments for panels A, B, and C. Error bars indicate SEM. The level of statistical significance for differences between Ag85B stimulation in the presence of IgG2a control Ab or agonistic anti-4-1BB mAb were determined by a two-tailed Student t test (*, p<0.05; **, p<0.01). In panel D, spleen cells after 2 days of culture and an overnight incubation with brefeldin A were stained with CD4-PE-Cy5 and then intracellular stained with PE anti-IL-10 Ab as reported in . Cells were then analyzed by flow cytometry. Dot plot were generated after gating on live CD4 + T lymphocytes and show frequency of IL-10-producing cells. In panel E, the IDO-inhibitor, 1-MT (400 µM), or neutralizing anti-TGF-β1.2 mAb (2 µg/ml) were added simultaneously to culture of spleen cells stimulated with Ag85B protein (5 µg/ml) in the presence of agonistic anti-4-1BB mAb (5 µg/ml) or rat IgG2a control Ab (5 µg/ml). Culture supernatants were harvested at 72 h for IFN-γ detection by specific quantitative sandwich ELISA Kits. Percent of 4-1BB-mediated inhibition of Ag85B-induced IFN-γ release is shown in round brackets.

Article Snippet: In some culture, the IDO-inhibitor, 1-methyltryptophan (1-MT) (Sigma-Aldrich, Saint Louis, MO) 400 µM or neutralizing anti-TGFβ1 mAb (clone 9016) (R&D System, Inc.) were also added.

Techniques: Magnetic Beads, Control, Sandwich ELISA, Two Tailed Test, Incubation, Staining, Flow Cytometry, Generated, Inhibition