Journal: The Journal of Experimental Medicine

Article Title: Interferon-α and Interleukin-12 Are Induced Differentially by Toll-like Receptor 7 Ligands in Human Blood Dendritic Cell Subsets

doi: 10.1084/jem.20020207

Figure Lengend Snippet: TLR-7 ligands induce the production of IL-12 and IFN-α from MDCs and PDCs, respectively. (A and B) After 24-h culture with various stimuli, concentration of IL-12 p40+p70 (A) and IFN-α (B) in the culture supernatants of MDCs, PDCs, and PBMCs were measured by ELISA. The data are shown by means ± SEM of five independent experiments. (C) After 5-h culture with R-848 (10 −6 M), intracellular staining of IL-12 and IFN-α in MDCs and PDCs, together with the staining of surface expression of CD40, was performed. Percentages of the respective cytokine producing DCs are indicated. This figure represents the results from one of three experiments.

Article Snippet: 10 μg/ml brefeldin A (Sigma-Aldrich) was added during the last 1 h. After the stimulation, MDCs and PDCs were stained with Cy-Chrome-labeled CD40 (5C3; BD PharMingen), and then fixed, permeabilized (FIX and PERM kit; Caltag Laboratories), and stained with FITC-labeled anti–IL-12 p40+p70 mAb (B-P24; DIACLONE Research) or unconjugated mouse anti–human IFN-α mAb (MC-16; Genzyme).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Expressing

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Schematic representation of ( A ) SARS-CoV-2 S pseudotyped lentivirus infection aided by S protein ACE2 binding and TMPRSS2. Knocking down of ACE2 and TMPRSS2 using LNP-siRNA LNP-trim inhibiting lentivirus entry ( B ). The presence of LNPs with surface ACE2 peptide (LNP-trap1), rhACE2 (LNP-trap2), and mAb (LNP-trap3) bind to the lentivirus, thereby inhibiting cell entry and infection ( C ).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Infection, Binding Assay

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Cytotoxicity of LNP-COOH analyzed using ( A ) cell impedance and ( B ) MTT assays in Calu-3 cells. Cytotoxicity MTT assays of LNP-PEP ( C ), LNP-rhACE2 ( D ), LNP-mAb ( E ), and LNP-si ACE2 ( F ) in Calu-3 cells. Values are expressed as the mean ± standard error of the mean (n = 3).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques:

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Quantitative determination of cellular accumulation of ( A ) LNP-PEP and ( B ) LNP-rhACE2 in Calu-3 cells over time. Representative confocal microscopy images from Calu-3 cells treated (3 hours) with cell-impermeable LNP-PEP ( C ‒ F ) and cell-permeable LNP- si ACE2 ( G ‒ J ). Plasma membrane from Calu-3 cells was stained with WGA Alexa Fluor TM 594 ( C and G ); LNP localization in Calu-3 cells was visualized with green fluorescent DiO dye ( D and H ); and cell nuclei were stained using Hoestch ( E and I ). Composite overlay of all three images showing cell membrane, nuclei, and LNPs ( F and J ). Orthogonal projections of Calu-3 cells treated with LNP-si ACE2 ( K ) for 3 hours. Two-dimensional images in z-position where red lines indicate cross-section through y-position and green lines indicate cross-section through x-position. The white dashed box in image K is enlarged. Values are expressed as the mean ± standard error of the mean (n = 3).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Confocal Microscopy, Membrane, Staining

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Relative ACE2 ( A ) and TMPRSS2( B ) mRNA levels in HEK-293-hACE2 cells at different time points after transfection with LNP-si ACE2 (40 nM si ACE2 ) and LNP-si TMPRSS2 (40 nM si TMPRSS2 ), respectively. The control cells in ( A ) and ( B ) received LNP-siSCR. Relative ACE2 mRNA level in Calu-3 cells 72 hours after receiving LNP-si ACE2 (40 nM si ACE2 ) against control cells that received LNP-siSCR ( C ). Relative ACE2 protein level in Calu-3 cells 72 hours after receiving LNP-si ACE2 (40 nM si ACE2 ) against control cells that received LNP-siSCR ( D ). The uncropped blots are shown in . Values are expressed as the mean ± standard error of the mean (n = 3). Statistical significance between the control and treatment groups were analyzed using t -test (**p < 0.01, ***p<0.001, ****p<0.0001; p < 0.05 was considered statistically significant).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Transfection

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Transduction efficiency (luciferase expression) of SARS-CoV-2 S pseudotyped luciferase lentivirus at the given MOIs in HEK-293 and HEK-293-hACE2 cell lines ( A ). Percentage inhibition of infection in ACE2 KD and TMPRSS2 KD HEK-293-hACE2 cells ( B ). Percentage inhibition of infection in ACE2 KD ( C ) and TMPRSS2 KD ( D ) in airway epithelial Calu-3 cell line at different siRNA concentrations (LNP-siRNA: 1, 2, 10, 20 and 40 nM; presented in the graph as log 10 values). The nonlinear regression curve fit analysis of siRNA concentration vs inhibition is presented in the graphs. Values are expressed as the mean ± standard error of the mean (n =3). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test (*p < 0.05, **p<0.01; p < 0.05 was considered statistically significant).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Transduction, Luciferase, Expressing, Inhibition, Infection, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Percentage inhibition of SARS-CoV-2 S pseudotyped luciferase lentivirus (MOI = 2) infection in Calu-3 cells in the presence of different concentrations (Log ( C ) of ACE2 peptide and LNP-PEP ( A ), rhACE2 and LNP-rhACE2 ( B ), mAb and LNP-mAb ( C ). The nonlinear regression curve fit analysis of concentration vs inhibition is presented in the graphs. Values are expressed as the mean ± standard error of the mean (n = 3).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Inhibition, Luciferase, Infection, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Percentage inhibition of SARS-Cov-2 S pseudotyped luciferase lentivirus infection (MOI = 2) in airway epithelial Calu-3 cell line following a combination of conditions including LNP-PEP treatment and ACE2 and TMPRSS2 knockdown. The extent of infection was measured following a luciferase assay. Values are expressed as the mean ± standard error of the mean (n = 3). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test (*p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001; p < 0.05 was considered statistically significant).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Inhibition, Luciferase, Infection

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Percentage inhibition of SARS-Cov-2 S pseudotyped luciferase LV infection (MOI = 2) in airway epithelial Calu-3 cell line following treatment with different concentrations of lactoferrin ( A ), camostat mesylate ( B ), and carrageenan ( C ). Percentage inhibition of infection in Calu-3 cells with various combinatory treatments of LNP-PEP (0.1 µg/mL), lactoferrin (10 µM), camostat mesylate (1 µM), and carrageenan (1 µM) ( D ). The extent of infection was measured following a luciferase assay. Values are expressed as the mean ± standard error of the mean (n = 3). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test (*p < 0.05, ****p<0.0001; p < 0.05 was considered statistically significant).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Inhibition, Luciferase, Infection

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Distribution of LNP-PEP ( A ) and LNP-si ACE2 ( B ) at different time points up to 24 hours after intranasal administration in nude mice. The LNPs were tagged with IR dye (DiR) to aid visualization. The NIR fluorescence images (Ex/Em 745/800) were acquired and analyzed using IVIS imager. The quantitative analysis of fluorescent LNP-PEP in the head ( C ) and body ( D ) regions at different time points. The quantitative analysis of fluorescent LNP-si ACE2 in the head ( E ) and body ( F ) regions at different time points. Values are expressed as the mean ± standard error of the mean (n = 4).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Fluorescence

Journal: Pharmaceutical Biology

Article Title: Tanshinone IIA alleviates ovalbumin-induced allergic rhinitis symptoms by inhibiting Th2 cytokine production and mast cell histamine release in mice

doi: 10.1080/13880209.2022.2034894

Figure Lengend Snippet: TIIA at 5 and 10 μmol/L had no effect on the viability of human mast cells, and reversed the promotion of histamine release and mast cell degranulation induced by C48/80. (A) The viability of human mast cell line HMC-1 cells after being treated with TIIA was examined by MTT assay. (B) Histamine content of human mast cell line HMC-1 cells after treatment with C48/80 and TIIA in culture medium (cell-free supernatants) and total suspensions was determined by ELISA. (C) The degranulation of human mast cell line HMC-1 cells after being treated with C48/80 and TIIA was detected by toluidine blue staining. *** p < 0.005, ### p < 0.005, * vs. control; # vs. OVA. TIIA: tanshinone IIA; MTT: methyl tetrazolium; ELISA: enzyme-linked immunosorbent assay; C48/80: compound 48/80.

Article Snippet: Mice serum was collected, and the concentrations of OVA-IgE and OVA-immunoglobulin G1 (IgG1) in mice serum were measured according to the instructions of mice OVA-IgE enzyme-linked immunosorbent assay (ELISA) kit (F10731, Westang, Shanghai, China) and mice OVA-IgG1 ELISA kit (3013, Chondrex, Woodinville, WA), respectively.

Techniques: MTT Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Foods

Article Title: Unripe Black Raspberry ( Rubus coreanus Miquel) Extract and Its Constitute, Ellagic Acid Induces T Cell Activation and Antitumor Immunity by Blocking PD-1/PD-L1 Interaction

doi: 10.3390/foods9111590

Figure Lengend Snippet: Effect of Rubus coreanus extract (RCE) on programmed cell death protein 1 (PD-1)/PD-1 ligand-1 (PD-L1) protein interaction using competitive enzyme-linked immunosorbent assay (ELISA) or cell-based assay. ( A , B ) The effect of RCE on the binding of PD-1 and PD-L1 in vitro using a competitive ELISA assay. ( C , D ) The effect of RCE on cell viability of PD-1/NFAT Jurkat cells ( C ) and PD-L1/aAPC CHO-K1 cells ( E ) using the Cell Counting Kit-8 (CCK) assay. Each of the cells were treated according to the indicated concentrations of RCE for 24 h. ( E ) The effect of RCE on cellular PD-1/PD-L1 blockade activity. PD-1/NFAT Jurkat effector cells and PD-L1/aAPC CHO-K1 target cells were co-cultured with RCE or αPD-L1 for 24 h at the indicated concentrations. The relative PD-1/PD-L1 blockade activity were measured by PD-1/NFAT luciferase reporter assay. ( F ) Effect of RCE on IL-2 cytokine release in co-cultured cell model. Cell culture media was analyzed to measure IL-2 level by cytokine ELISA. Data are presented as mean ± S.E. of three representative independent experiments. Asterisks indicate significant inhibition of PD-1/PD-L1 binding activity by each test inhibitor as compared with the vehicle-treated group; *** p < 0.001, ** p < 0.01, * p < 0.05, compared with the vehicle group.

Article Snippet: The human PD-1/PD-L1 competitive enzyme-linked immunosorbent assay (ELISA) kit and antagonist antibody to human PD-L1 (αPD-L1) were purchased from BPS Bioscience Inc. (San Diego, CA, USA).

Techniques: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Cell Based Assay, Binding Assay, In Vitro, Cell Counting, Activity Assay, Cell Culture, Luciferase, Reporter Assay, Inhibition