Journal: Frontiers in Immunology

Article Title: Combined p14ARF and Interferon-β Gene Transfer to the Human Melanoma Cell Line SK-MEL-147 Promotes Oncolysis and Immune Activation

doi: 10.3389/fimmu.2020.576658

Figure Lengend Snippet: Emission of immunogenic cell death markers induced by combined adenoviral p14ARF + IFNβ gene transfer. SK-MEL-147 cells transduced as previously described, incubated for 48h h before cells and supernatants were collected for ICD assays. (A) Calreticulin exposure was assessed by flow cytometry after specific antibody staining. Representative dot plots and a bar graph showing the mean and standard deviation from three independent tests with three technical replicates each. (B) Supernatant from the same cultures were collected and evaluated for ATP secretion using a luciferase-based assay (RLU, relative light units). Data represent the mean and standard deviation from at least three independent experiments. (C) Detection of secreted IFNβ protein from cell supernatant by ELISA. Data represent the mean and standard deviation from at least three independent experiments. For both (A–C) , statistical analyses were performed using one-way ANOVA test followed by the Bonferroni post-test. *p < 0.05, **p < 0.005, and ***p < 0.0005.

Article Snippet: For the evaluation of calreticulin exposure through flow cytometry, cells were probed with rabbit anti-calreticulin antibody (Novus Biologicals, Littleton, CO, USA), followed by the Alexa488-conjugated anti-rabbit secondary antibody (Thermo Fisher Scientific).

Techniques: Incubation, Flow Cytometry, Staining, Standard Deviation, Luciferase, Enzyme-linked Immunosorbent Assay