Computational protocol: The Transmembrane Domains of TNF-Related Apoptosis-Inducing Ligand (TRAIL) Receptors 1 and 2 Co-Regulate Apoptotic Signaling Capacity

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Protocol publication

[…] MF expressing the respective chimeric receptor (2×105 cells) were seeded on 18 mm cover slips and cultured overnight. The following day, cells were transiently transfected with pEGFP-FADD plasmide using Lipofectamine™ 2000 (Invitrogen Life Technologies GmbH, Darmstadt, Germany) according to the manufacturer's instructions. 24 hours after transfection, cells were stimulated with antibody-crosslinked TRAIL (300 ng/ml sTRAIL crosslinked with 2 µg/ml anti-FLAG-M2 antibody (Sigma Aldrich), or 100 ng/ml Alexa Fluor 546-coupled recombinant human TNF. Subsequently, cells were washed (3×5 min, PBS), fixed (4% paraformaldehyde in PBS; 15 min at RT) and blocked (5% FCS, 0.05% Tween-20 in PBS, 30 min at RT). TRAIL stimulated cells were additionally immunostained with 1 µg/ml anti-mouse IgG Alexa Fluor® 546 (Molecular Probes, Invitrogen). Excess antibody and ligand was removed by washing with PBS, and coverslips were mounted on microscope slides using Fluoromount G™ (SouthernBiotech, Birmingham, USA), before being analyzed by confocal microscopy (Leica TCS confocal laser scanning microscope (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany) or Zeiss LSM710 microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany)). Colocalization analysis was performed using the ImageJ plug-in JACoP . Pearson's correlation coefficient was calculated from optical sections of confocal z-stacks. Costes' method for automatic thresholding and testing of statistical significance of colocalization was applied . Statistical analysis was performed using GraphPad Prism4. […]

Pipeline specifications

Software tools Coloc, ImageJ, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Neoplasms