hekblue detection medium  (InvivoGen)

Journal: International journal of molecular sciences

Article Title: Necrotic Cells from Head and Neck Carcinomas Release Biomolecules That Are Activating Toll-like Receptor 3.

doi: 10.3390/ijms242015269

Figure Lengend Snippet: Figure 1. Flow chart showing the experimental design of the study. Conditioned media were collected from cells stressed with irradiation, serum starvation, hypoxia and oxidative stress. In addition, extracellular vesicles were isolated from conditioned media of irradiated cells. Both crude conditioned media and isolated extracellular vesicles were used to stimulate HEKBlue cells in order to measure the activation of the TLR3 signaling pathway (A). Additionally, necrotic fluids were collected from tumor grafts carried by nude or Balb/C mice (B) and from tumor tissues of patients bearing head and neck carcinomas (C). These fluids were also tested on HEKBlue cells in order to measure the activation of the TLR3 signaling pathway.

Article Snippet: Cells were grown to 50–70% confluence and plated at a cell density of 3 × 104 viable cells in 150 μL per well in a 96-well flat-bottomed tissue culture plate prior to treatment in HEKBlue detection medium (Invivogen).

Techniques: Irradiation, Isolation, Activation Assay

Journal: International journal of molecular sciences

Article Title: Necrotic Cells from Head and Neck Carcinomas Release Biomolecules That Are Activating Toll-like Receptor 3.

doi: 10.3390/ijms242015269

Figure Lengend Snippet: Figure 2. TLR3 activation in HEKBlue-TLR3 and HEKBlue-null reporter cells after treatment with supernatants from Detroit 562 (A), FaDu (B) and SQ20B cells (C) stressed with irradiation, H2O2, serum deprivation and hypoxia. Ctrl: cells were treated only with fresh culture medium; HY: hypoxia treatment. * p < 0.05, ** p < 0.01, ## p < 0.01 (HEKBlue-TLR3 in relation to HEKBlue-null control), + p < 0.05. (HEKBlue-null in relation to HEKBlue-null control). Statistical significance was determined by t-test.

Article Snippet: Cells were grown to 50–70% confluence and plated at a cell density of 3 × 104 viable cells in 150 μL per well in a 96-well flat-bottomed tissue culture plate prior to treatment in HEKBlue detection medium (Invivogen).

Techniques: Activation Assay, Irradiation, Control

Journal: International journal of molecular sciences

Article Title: Necrotic Cells from Head and Neck Carcinomas Release Biomolecules That Are Activating Toll-like Receptor 3.

doi: 10.3390/ijms242015269

Figure Lengend Snippet: Figure 4. Exosome characterization and the treatment of HEKBlue-TLR3 reporter cells with exosomes. Transmission electron microscopy (TEM) images of exosomes derived from Detroit 562, FaDu and SQ20B cell supernatants from control non-irradiated cells and cells after irradiation with 10 Gy (A). Western blot of proteins isolated from exosomes (B) and cell lysate. The loading control is the membrane stained with amido black (C). TLR3 activation after the treatment of HEKBlue-TLR3 cells with exosomes (800 ng) isolated from control non-irradiated cells and cells after irradiation with 10 Gy (D). Comparison between TLR3 activation with crude medium and exosomes isolated after irradiation (E). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were grown to 50–70% confluence and plated at a cell density of 3 × 104 viable cells in 150 μL per well in a 96-well flat-bottomed tissue culture plate prior to treatment in HEKBlue detection medium (Invivogen).

Techniques: Transmission Assay, Electron Microscopy, Derivative Assay, Control, Irradiation, Western Blot, Isolation, Membrane, Staining, Activation Assay, Comparison

Journal: International journal of molecular sciences

Article Title: Necrotic Cells from Head and Neck Carcinomas Release Biomolecules That Are Activating Toll-like Receptor 3.

doi: 10.3390/ijms242015269

Figure Lengend Snippet: Figure 5. TLR3 activation after the treatment of HEKBlue-TLR3 and HEKBlue-null cells with necrotic fluids derived from oral squamous cell carcinoma patients: patient 1, patient 2, patient 3, patient 4 and patient 5. Ctrl—cells were treated only with fresh culture medium; RNAse—sample pretreated with RNAse; TLR3ab—sample treated with monoclonal TLR3 antibody; pIC—cells treated with 100 ng/mL poly(I:C). The amount of RNA and protein in each sample is also presented. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001 (HEKBlue-TLR3 in relation to HEKBlue-null control). + p < 0.05 (HEKBlue-null patient in relation to HEKBlue-null control), $ p < 0.05 (HEKBlue-null treatment in relation to HEKBlue-null patient).

Article Snippet: Cells were grown to 50–70% confluence and plated at a cell density of 3 × 104 viable cells in 150 μL per well in a 96-well flat-bottomed tissue culture plate prior to treatment in HEKBlue detection medium (Invivogen).

Techniques: Activation Assay, Derivative Assay, Control

Journal: International journal of molecular sciences

Article Title: Necrotic Cells from Head and Neck Carcinomas Release Biomolecules That Are Activating Toll-like Receptor 3.

doi: 10.3390/ijms242015269

Figure Lengend Snippet: Figure 6. TLR3 activation after the treatment of HEKBlue-TLR3 and HEKBlue-null cells with necrotic fluids derived from different mice models: C18, C17 and Renca. Ctrl—cells treated only with medium; RNAse—sample pretreated with RNAse; TLR3ab—sample treated with monoclonal TLR3 antibody; pIC—cells treated with 100 ng/mL poly(I:C). The amount of RNA and protein in each sample is also presented. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ## p < 0.01, ### p < 0.001 (HEKBlue-TLR3 in relation to HEKBlue-null control).

Article Snippet: Cells were grown to 50–70% confluence and plated at a cell density of 3 × 104 viable cells in 150 μL per well in a 96-well flat-bottomed tissue culture plate prior to treatment in HEKBlue detection medium (Invivogen).

Techniques: Activation Assay, Derivative Assay, Control