Journal: Cell

Article Title: Serum Amyloid A Proteins Induce Pathogenic T H 17 Cells and Promote Inflammatory Disease

doi: 10.1016/j.cell.2019.11.026

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: Flow Cytometry: anti-mouse MHCII (M5/114.15.2) PerCP-cy5.5 , BD Bioscience , Cat. 562363.

Techniques: Flow Cytometry, Western Blot, In Vitro, Cell Differentiation, Activation Assay, In Vivo, Immunohistochemistry, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, SYBR Green Assay, Ex Vivo, Isolation, Knock-In, Knock-Out, Software

Journal: Theranostics

Article Title: Engineering blood exosomes for tumor-targeting efficient gene/chemo combination therapy

doi: 10.7150/thno.45028

Figure Lengend Snippet: Characterization of D-Exos/miR21i-L17E. (A, B) The particle size distribution and representative TEM images of SMNC-Exos and D-Exos/miR21i-L17E, respectively. (scale bar: 20 nm) (C) Western blots analysis of specific Tf receptors and exosome marker proteins (CD63, CD81 and CD9) of SMNC-Exos, D-Exos/miR21i and D-Exos/miR21i-L17E. (D) Detection of fluorescent signals in purified SMNC-Exos, D-Exos, D-Exos/miR21i and D-Exos/miR21i-L17E at different excitation/emission (Ex/Em) wavelength. The SMNC-Exos and chol-miR21i contained in each sample were labeled by FITC and Cy5, respectively. (E) UV-vis spectra of the SMNC-Exos, Dox, chol-miR21i-Cy5, L17E and D-Exos/miR21i-L17E. The inset shows the magnified UV-vis spectra from 600 to 700 nm. (F) Gel retention assay of D-Exos/miR21i prepared with D-Exos and varied concentrations of cholesterol-conjugated miR-21i or non-cholesterol-conjugated miR-21i. The Cy5-labeled miR-21i bands are displayed. (G) Schematic of the methods for confirming the localization of chol-miR21i on D-Exos/miR21i. Cy5-labeled miR-21 is complementary to chol-miR21i on D-Exos/miR21i. If miR-21i is completely display on the surface of exosomes, Cy5-miR21 will bind to D-Exos/miR21i, otherwise it will not. (H) Analysis for the binding of Cy5-miR21 to D-Exos/miR21i by gel electrophoresis. The Cy5-labeled miR-21 bands are displayed here. (I) Release profiles of Dox from D-Exos/miR21i-L17E in PBS (pH=7.4) and acetate buffer (pH=5.0) for 24 h. Data in (I) are presented as the mean ± standard deviation (SD) from three independent experiments (n=3).

Article Snippet: Cy5 and Cy5.5 fluorescence dyes were purchased from US Everbright ® Inc (Suzhou, China).

Techniques: Western Blot, Marker, Purification, Labeling, Binding Assay, Nucleic Acid Electrophoresis, Standard Deviation

Journal: Theranostics

Article Title: Engineering blood exosomes for tumor-targeting efficient gene/chemo combination therapy

doi: 10.7150/thno.45028

Figure Lengend Snippet: Cellular uptake and intracellular distribution of D-Exos/miR21i-L17E. (A) Confocal images of U87 cells incubated with D-Exos/miR21i and D-Exos/miR21i-L17E for different times (1 h, 2 h, 4 h). The fluorescence spots of Dox (red) and chol-miR21i-Cy5 (purple) were observed. The nucleus and endo/lysosome were stained with DAPI (blue) and Lysotracker Green (green), respectively. (B) Co-localization ratio of the miR-21i signals (purple) in D-Exos/miR21i and D-Exos/miR21i-L17E to endosome signals (green) at different time points. (C) Flow cytometry profile of U87 cells incubated with free Dox and chol-miR21i, D-Exos, Exos/miR21i, D-Exos/miR21i and D-Exos/miR21i-L17E for 4 h. The y-axis represents cell counts and the x-axis represents the Dox and chol-miR21i-Cy5 fluorescence intensity, respectively. Data in (B) are presented as the mean ± SD (n>50) from three independent experiments (n=3). The significant levels are shown as ** p < 0.01, *** p < 0.001.

Article Snippet: Cy5 and Cy5.5 fluorescence dyes were purchased from US Everbright ® Inc (Suzhou, China).

Techniques: Incubation, Fluorescence, Staining, Flow Cytometry

Journal: Theranostics

Article Title: Engineering blood exosomes for tumor-targeting efficient gene/chemo combination therapy

doi: 10.7150/thno.45028

Figure Lengend Snippet: Tumor targeting efficiency of engineered exosomes in tumor-bearing mice in vivo . (A) In vivo NIRF imaging of U87 tumor-bearing nude mice after intravenous injection of Cy5.5-labeled D-Exos/miR21i-L17E with/without an external MF at 1 h, 4 h and 24 h post-injection. (B) Quantitative analysis of the tumor accumulation of the D-Exos/miR21i-L17E based on the fluorescence intensity from the in vivo images. (C) The accumulation of Dox and Cy5-miR21i in the tumor section, where red represents Dox, green represents miR21i and cell nucleus was stained by DAPI. The scale bar is 100 μm. Data in (B) are presented as the mean ± SD (n=5).

Article Snippet: Cy5 and Cy5.5 fluorescence dyes were purchased from US Everbright ® Inc (Suzhou, China).

Techniques: In Vivo, Imaging, Injection, Labeling, Fluorescence, Staining

Journal: PLoS ONE

Article Title: The novel multi-cytokine inhibitor TO-207 specifically inhibits pro-inflammatory cytokine secretion in monocytes without affecting the killing ability of CAR T cells

doi: 10.1371/journal.pone.0231896

Figure Lengend Snippet: A) Effects of PSL and TO-207 on the cytotoxicity of CAR T cells. K562/CD19/fLucEGFP cells (3 × 10 3 ) were co-cultured with CD4 + or CD8 + CAR T cells (1.5 × 10 4 ), and treated with different concentrations of PSL and TO-207. Viable target cells were quantified using the luciferase assay after 72 h of co-culture. Values were normalized to viable K562/CD19/fLucEGFP cells cultured alone. The error bars represent SDs from three independent experiments. B) Effects of PSL and TO-207 on CAR T cell degranulation. K562/CD19 cells (5 × 10 5 ) and CAR T cells (5 × 10 5 ) were co-cultured in 500 μl T-cell expansion medium in the absence or presence of PSL or TO-207. Following 68 h of co-culture, monensin (2 μM) and APC–anti-human CD107a antibody were added. Cells were incubated for an additional 4 h, and membrane expression of CD107a was determined by flow cytometry. Representative data of four independent experiments are shown. C) Mean fluorescence intensity (MFI) of samples in Fig 5B. The error bars represent SEs from four independent experiments. D) Sustained cytotoxicity of CAR T cells following TO-207 treatment. K562/CD19/fLucEGFP cells (1 × 10 4 ), CAR T cells (5 × 10 4 ), and CD14 + cells (5 × 10 4 ) were co-cultured in the absence or presence of PSL or TO-207 for 72 h, and viable target cells were quantified by the luciferase assay. Values were normalized to the well containing K562/CD19/fLucEGFP cells alone. The error bars represent SDs from three independent experiments. The linear dose-response relationship was assessed using log-transformed dose values (to the base 10) in a mixed model, in which the zero dose was replaced by the log (minimal dose) - 1. P < 0.05 was considered statistically significant. n.s.: not significant.

Article Snippet: Flow cytometry was performed using allophycocyanin (APC)-conjugated IgG 1 (BioLegend), APC–anti-human CD19 antibody (BioLegend), APC–anti human CD107a (BioLegend), APC–Cy7–anti-human CD3 antibody (BioLegend), V500–anti-human CD4 antibody (BD Biosciences, San Jose, CA, USA), and PerCP–Cy5.5–anti-human CD8 antibody (TONBO Biosciences, San Diego, CA, USA).

Techniques: Cell Culture, Luciferase, Co-Culture Assay, Incubation, Membrane, Expressing, Flow Cytometry, Fluorescence, Transformation Assay

Journal: Scientific Reports

Article Title: PLGA nanoparticles co-delivering MDR1 and BCL2 siRNA for overcoming resistance of paclitaxel and cisplatin in recurrent or advanced ovarian cancer

doi: 10.1038/s41598-018-25930-7

Figure Lengend Snippet: Efficient cellular internalization of siRNA@PLGA NPs on MDR ovarian cancer cells. ( A ) Flow cytometric histogram showing cellular internalization of the fluorescent siRNA@PLGA NPs on SKOV3-TR and A2780-CP20 cancer cells respectively. The resistance cell lines were treated with fluorescent Cy5.5-siRNA@PLGA NPs, and 24 h later, sorted. A prefixed gate region was allowed only for Cy5.5 fluorescence. The untreated cells are indicated as a control. ( B ) Z-stack confocal microscopic images of SKOV3-TR and A2780-CP20 cancer cells treated with Cy5.5-labeled siRNA@PLGA NPs. Cy5.5-siRNA and DAPI dyes are represented by red and blue, respectively. Scale bar: 10 μm.

Article Snippet: For flow cytometry analysis, each cell lines treated with the fluorescent Cy5.5-siRNA-loaded PLGA NPs were examined by Guava EasyCyte flow cytometer (Merck Millipore) equipped with 640 nm laser.

Techniques: Fluorescence, Labeling