Journal: Oncotarget

Article Title: Pancreatic adenocarcinoma upregulated factor serves as adjuvant by activating dendritic cells through stimulation of TLR4

doi:

Figure Lengend Snippet: A. DCs activation was demonstrated by measuring pro-inflammatory cytokines and type I interferon, DCs activation factors, in culture supernatant of DCs incubated with PAUF (1 μg or 10 μg) or LPS (100 ng) as a positive control for 16 hr with ELISA. B. and C. To probe maturation level of DCs, after DCs were incubated with PAUF or LPS for 16 hr, maturation surface markers (CD40, CD80, CD86, MHC class I) were confirmed by using flow cytometry analysis after incubation with PAUF or LPS as a positive control and PBS as a negative control. D. To determine MAPKs and IkB-α, intracellular protein marker of activated DCs, DCs were treated with PAUF (5 μg) of time course and analyzed by using Western Blot analysis. **: P < 0.01.

Article Snippet: FITC-conjugated anti-mouse CD11c antibody, PE-conjugated anti-mouse CD8a, CD80, CD86, MHC class I antibody and Mouse IFN-β ELISA kit were purchased from Biolegend (San Diego, CA).

Techniques: Activation Assay, Incubation, Positive Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Negative Control, Marker, Western Blot

Journal: Oncotarget

Article Title: Pancreatic adenocarcinoma upregulated factor serves as adjuvant by activating dendritic cells through stimulation of TLR4

doi:

Figure Lengend Snippet: A. To confirm maturation factor (CD80, CD83 and CD86) and migration factor (CCR7) of mature DCs compared with immature DCs (imDC), DCs (PAUF-DC1, PAUF-DC2, and PAUF-DC3) were treated with PAUF of various concentrations (5 μg, 10 μg and 20 μg/ml, respectively) or LPS (100 ng/ml; LPS-DC) for 2 days, and then analyzed by flow cytometry. Histogram shows antibody staining (in dark) relative to isotype-matched control (transparent). Data are mean fluorescence intensities (MFI) of three independent experiments. B. The bar graph depicts mean fluorescence intensity (MFI) of each surface markers. C. Cytokines (IL-12p70, IL-10 and IL-23) in the culture supernatant of PAUF (5 μg, 10 μg and 20 μg/ml) of various concentration or LPS (100 ng/ml)-treated DCs for 2 days were determined by ELISA. Immature DC culture supernatant was used as a control. **: P < 0.01.

Article Snippet: FITC-conjugated anti-mouse CD11c antibody, PE-conjugated anti-mouse CD8a, CD80, CD86, MHC class I antibody and Mouse IFN-β ELISA kit were purchased from Biolegend (San Diego, CA).

Techniques: Migration, Flow Cytometry, Staining, Control, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: BAY 60-6583 enhances cytokine secretion of activated anti-CD133 CAR T cells. Anti-CD133 CAR T cells were co-cultured with U251-CD133OE cells, the effector-to-target cell (E:T) ratio was 4:1. Vehicle and eight small molecules targeting adenosine receptors were added at different final concentrations. After 16 h, the medium was collected and secretion of INF-γ (A) and GM-CSF (B) was detected using AlphaLISA kits. Data were normalized to vehicle control and are represented as the mean ± SD of triplicates from a representative experiment of n = 3 experiments.

Article Snippet: After 16 h, the supernatants of the cultures were collected, and cytokines (TNF-α, IFN-γ, and GM-CSF) were detected using AlphaLISA detection kits (TNF-α, AL208C; IFN-γ, AL217C; and GM-CSF, AL216C) from PerkinElmer, Waltham, MA, United States.

Techniques: Cell Culture, Control

Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: BAY 60-6583-mediated CAR T cell enhancement requires T cell activation. Anti-CD133 CAR T cells (1 × 10 6 ) were activated by co-incubation with 2.5 × 10 5 U251-CD133OE cells (A) or CD3/CD28 beads (B) ; unstimulated CAR T cells (C) were used as control. Vehicle and BAY 60-6583 were added at different final concentrations. After 16 h, the medium was collected and the secretion of GM-CSF, INF-γ, and TNF-α was detected using AlphaLISA kits. Data were normalized to vehicle control and are represented as the mean ± SD of triplicates from a representative experiment of n = 4 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by 1-way ANOVA; ns, not significant.

Article Snippet: After 16 h, the supernatants of the cultures were collected, and cytokines (TNF-α, IFN-γ, and GM-CSF) were detected using AlphaLISA detection kits (TNF-α, AL208C; IFN-γ, AL217C; and GM-CSF, AL216C) from PerkinElmer, Waltham, MA, United States.

Techniques: Activation Assay, Incubation, Control

Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: BAY 60-6583 specifically improved activities of CD133-CAR T cells. (A–D) Anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells or U251 WT luc cells at different E:T ratios. Co-cultures were performed in the presence of vehicle or BAY 60-6583. After 16 h, the medium was collected, and the secretion of INF-γ (A) , TNF-α (B) , and GM-CSF (C) was detected using AlphaLISA kits (results for the E:T ratio of 4:1 are shown). (D) At 48 h after co-culture, cytotoxicity was determined by detecting the bioluminescence signal. Data are represented as the mean ± SD of triplicates from a representative experiment of n = 5 experiments. **** p < 0.0001 by 2-way ANOVA; ns, not significant (E) CFSE-labeled CAR T cells were co-cultured with irradiated U251-CD133OE cells in the presence of BAY 60-6583 or vehicle control. After 120 h, flow cytometry was used to analyze cell proliferation. Results shown are from a representative experiment of n = 4 experiments.

Article Snippet: After 16 h, the supernatants of the cultures were collected, and cytokines (TNF-α, IFN-γ, and GM-CSF) were detected using AlphaLISA detection kits (TNF-α, AL208C; IFN-γ, AL217C; and GM-CSF, AL216C) from PerkinElmer, Waltham, MA, United States.

Techniques: Cell Culture, Co-Culture Assay, Labeling, Irradiation, Control, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: The photo-affinity probe enhanced CAR T cell effects similarly to unmodified BAY 60-6583 (A) The structure of the photo-affinity probe (B–D) Anti-HER2 CAR T cells (1 × 10 5 ) were activated by TransAct (B) or 4 × 10 5 MDA-MB-453 luc cells (C, D) . Probe, BAY 60-6583, or vehicle control was added at the beginning. After 16 h, the medium was collected, and the secretion of INF-γ, TNF-α, and GM-CSF was detected using AlphaLISA kits (B, C) ; 48 h later, cytotoxicity was assessed (D) . Data are represented as the mean ± SD from a representative experiment of n = 3 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by 1-way ANOVA; ns, not significant.

Article Snippet: After 16 h, the supernatants of the cultures were collected, and cytokines (TNF-α, IFN-γ, and GM-CSF) were detected using AlphaLISA detection kits (TNF-α, AL208C; IFN-γ, AL217C; and GM-CSF, AL216C) from PerkinElmer, Waltham, MA, United States.

Techniques: Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Active Fraction Combination from Liuwei Dihuang Decoction (LW-AFC) Alleviated the LPS-Induced Long-Term Potentiation Impairment and Glial Cells Activation in Hippocampus of Mice by Modulating Immune Responses

doi: 10.1155/2019/3040972

Figure Lengend Snippet: Effect of LW-AFC on cytokine secretion in the (a) hippocampus and (b) plasma of LPS-treated mice. ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 vs. con and Student's t -test; ### p < 0.001, ## p < 0.01, and # p < 0.05 vs . mod, one-way ANOVA, and Dunnett's test, mean ± SD, n = 5–8. A, IL-1 β ; B, IL-6; C, MCP-1; D, G-CSF; E, IL-10; F, TNF- α ; G, IL-6; H, IFN γ ; I, IL-12; J, IL-10. con, control; mod, model; INDO, indomethacin.

Article Snippet: The secretion of TNF- α in the cell supernatant was determined using an AlphaLISA mouse TNF- α Kit (PerkinElmer, Waltham, MA, USA) according to the manufacturer's protocols using an EnVisionTM 2104 instrument (PerkinElmer, Waltham, MA, USA).

Techniques: Clinical Proteomics, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Active Fraction Combination from Liuwei Dihuang Decoction (LW-AFC) Alleviated the LPS-Induced Long-Term Potentiation Impairment and Glial Cells Activation in Hippocampus of Mice by Modulating Immune Responses

doi: 10.1155/2019/3040972

Figure Lengend Snippet: Effect of compounds from LW-AFC on cell viability and secretion of TNF- α in LPS-stimulated and non-LPS-stimulated BV-2 cells: (a) the cell viability of non-LPS-stimulated BV-2 cells; (b) the cell viability of LPS-stimulated BV-2 cells; (c) secretion of TNF- α in non-LPS-stimulated BV-2 cells; (d) secretion of TNF- α in LPS-stimulated BV-2 cells. ∗∗∗ p < 0.001 and ∗∗ p < 0.01 vs . con and Student's t -test; ### p < 0.001, ## p < 0.01, and # p < 0.05, vs . mod, one-way ANOVA, and Dunnett's test. Mean ± SD n = 3. con, control; mod, model; INDO, indomethacin; DEX, dexamethasone.

Article Snippet: The secretion of TNF- α in the cell supernatant was determined using an AlphaLISA mouse TNF- α Kit (PerkinElmer, Waltham, MA, USA) according to the manufacturer's protocols using an EnVisionTM 2104 instrument (PerkinElmer, Waltham, MA, USA).

Techniques: Control

Journal: Biochemistry

Article Title: Glucagon-like Peptide 1 Conjugated to Recombinant Human Serum Albumin Variants with Modified Neonatal Fc Receptor Binding Properties. Impact on Molecular Structure and Half-Life.

doi: 10.1021/acs.biochem.7b00492

Figure Lengend Snippet: Figure 7 (A): The structure of rHSA in complex with the extracellular part of FcRn (pdbid: 4N0F) superimposed on a SAXS-based BUNCH model of GLP-1-PEG-HB-albumin. HSA is displayed in gray (domain I: medium gray, domain II: dark gray, domain III: light gray), FcRn α in blue and β-2m in green and the conjugated GLP-1 in pink. (B): Turned and seen from the top.

Article Snippet: Serum concentrations were determined using an AlphaLISA method employing a commercial HSA kit (AL296C, Perkin Elmer, MA, USA) combined with a monoclonal anti-GLP-1 biotinylated monoclonal antibody (Ab121072, Abcam) to detect both albumin and GLP1 moieties simultaneously; for albumin alone, the albumin kit was used.

Techniques:

Journal: Biochemistry

Article Title: Glucagon-like Peptide 1 Conjugated to Recombinant Human Serum Albumin Variants with Modified Neonatal Fc Receptor Binding Properties. Impact on Molecular Structure and Half-Life.

doi: 10.1021/acs.biochem.7b00492

Figure Lengend Snippet: Figure 8 (A): The structure of GLP-1 (turquoise) in complex with the extracellular part of the GLP-1 receptor (blue), (pdbid: 3IOL) superimposed on a SAXS-based BUNCH model of GLP-1-PEG-HB-albumin. The conjugated GLP-1 is shown in pink, and HSA is displayed in gray (domain I: medium gray, domain II: dark gray, domain III: light gray). (B): A rotated version of (A), but with the full-length GLP-1R crystal structure38 (in bronze) (pdbid: 5NX2) superimposed, and a membrane is indicated as a light blue shadow.

Article Snippet: Serum concentrations were determined using an AlphaLISA method employing a commercial HSA kit (AL296C, Perkin Elmer, MA, USA) combined with a monoclonal anti-GLP-1 biotinylated monoclonal antibody (Ab121072, Abcam) to detect both albumin and GLP1 moieties simultaneously; for albumin alone, the albumin kit was used.

Techniques: Membrane