anti human pe conjugated antibody against trail r2  (R&D Systems)

Journal: Cells

Article Title: Enhanced Anti-Melanoma Activity of Nutlin-3a Delivered via Ethosomes: Targeting p53-Mediated Apoptosis in HT144 Cells

doi: 10.3390/cells13201678

Figure Lengend Snippet: Nutlin-3a-loaded ethosomes upregulate surface death receptors TRAIL-R2 of p53 wild-type melanoma cells. Surface cytofluorimetric analysis of HT144 expressing p53 wild-type ( A ) and SK-MEL-28 expressing p53 mut ( B ) cell cultures treated with nutlin-3a or nutlin-3a-loaded ethosomes with equivalent nutlin-3a concentrations after 24 h. Vehicle and empty ethosomes are reported as control vehicles. Data are calculated as the fold increase in median fluorescence values with respect to untreated cells (set to 1). Bars represent the mean ± SEM, and each circle denotes the value of each experimental replicate. Statistical analysis was performed by means of ANOVA followed by Bonferroni’s post hoc test. *** indicates a p -value ≤ 0.001.

Article Snippet: Cells were treated for 48 h with nutlin-3a in solution, nutlin-3a-loaded ethosomes, and the vehicle and controls as described above, harvested, and then stained with anti-human PE-conjugated antibody against TRAIL-R2 (clone 71908, R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Control, Fluorescence

Journal: Molecular cancer research : MCR

Article Title: A genome-wide loss-of-function screen identifies SLC26A2 as a novel mediator of TRAIL resistance

doi: 10.1158/1541-7786.MCR-16-0234

Figure Lengend Snippet: (A) Flow cytometry analysis in BJAB-LEXR cells with stable, lentiviral, shRNAs against SLC26A2 or a Non-targeting shRNA after 24hrs of treatment with TRAIL (20ng/mL) to assess propidium iodide (PI) positive cells as an indication of dead or late apoptotic cells. Representative experiment is shown (of 2 experiments). (B) MDA-231 SENS and TRAILR cells plated in replicates of 6 were treated with TRAIL (100ng/mL) and a green fluoregenic substrate for activated-caspase3/7. Green fluorescence was measured every 2 hours with 100ng/mL TRAIL by light microscopy using real time in-vitro imaging. Statistical significance assessed by 2-way ANOVA with a Tukey post-test. A representative figure is shown (of 2 experiments). (C) Triplicate samples of 231 SEN and 231 TRAILR were analyzed for RNA expression of SLC26A2, DR4, and DR5, using microarray analysis. The expression is presented as a heat map relative to an average expression of a combination of constitutive housekeeping genes. RNA and protein were isolated from 231 SEN cells and 231 TRAILR cells as well as 231 TRAILR cells transduced with non-targeting control shRNA (non-coding) or with either of 2 shRNA targeting SLC26A2 (SLC26A2 sh1 and SLC26A2 sh2, respectively). mRNA expression of (D) SLC26A2, (E) DR4 and (F) DR5 was determined by real time quantitative PCR. The expression is relative to the expression of GAPDH and was normalized to non-targeting shRNA vector-expressing cells. Representative experiments are shown (of ≥ 3 experiments) with each experiment performed in triplicate. Statistical significance assessed by 1-way ANOVA. Protein expression of these genes as well as β-actin was analyzed by Western blot as shown in (G). Surface expression of DR4 and DR5 was analyzed by flow cytometry as depicted in (H) and (I), respectively. Representative experiments are shown (of ≥ 3 experiments). Statistical significance assessed by 1-way ANOVA.

Article Snippet: 10μl/1X10 6 of PE conjugated antibodies against DR4 (FAB347P, R&D Systems) and DR5 (FAB6311P, R&D Systems) were then added and allowed to incubate for 30 minutes at room temperature in the dark followed by 3 washes with Flow Cytometry Staining Buffer.

Techniques: Flow Cytometry, shRNA, Fluorescence, Light Microscopy, In Vitro, Imaging, RNA Expression, Microarray, Expressing, Isolation, Transduction, Real-time Polymerase Chain Reaction, Plasmid Preparation, Western Blot