The Netherlands Cancer Institute

5 articles published in JoVE

• Biology

  
  Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling Dimitrios Laskaris*^1,2, Maria Azkanaz*^1,2, Mijke A. de Vreij-Kruidenier³, Doreen van Rijswoud-Ram³, Hendrik A. Messal^1,2, Jacco van Rheenen^{1,2 1}Department of Molecular Pathology, The Netherlands Cancer Institute, ²Oncode Institute, ³Animal Laboratory Facility, The Netherlands Cancer Institute Here, we present a method to obtain images of the intestine upon laser-induced wounding. By exposing the mouse intestine to a multiphoton laser, the loss of a single or multiple crypt(s) is induced locally. By repeatedly imaging the damaged area over months, the real-time dynamics of intestinal recovery is captured.

• Biochemistry

  
  Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy Jitske Bak¹, Lisa Landskron¹, Thijn R. Brummelkamp¹, Anastassis Perrakis^{1 1}Oncode Institute and Division of Biochemistry, Netherlands Cancer Institute Here, we describe a protocol to extract endogenous tubulin from mammalian cells, which can lack or contain specific microtubule-modifying enzymes, to obtain microtubules enriched for a specific modification. We then describe how the extracted microtubules can be decorated with purified microtubule-binding proteins to prepare grids for cryo-electron microscopy.

• Biochemistry

  
  Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery Misbha Ud Din Ahmad¹, Alexander Fish¹, Jeroen Molenaar¹, Sridhar Sreeramulu², Christian Richter², Nadide Altincekic², Harald Schwalbe², Hans Wienk¹, Anastassis Perrakis^{1 1}Oncode Institute and Division of Biochemistry, the Netherlands Cancer Institute, ²Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

• Cancer Research

  
  A Practical Guide for the Production and PET/CT Imaging of ⁶⁸Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice Else A. Aalbersberg¹, Martine M. Geluk-Jonker¹, Thirumaraichelvi Young-Mylvaganan¹, Linda J. de Wit-van der Veen¹, Marcel P. M. Stokkel^{1 1}Department of Nuclear Medicine, Netherlands Cancer Institute Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog ⁶⁸Ga-DOTATATE. This protocol details the radiolabeling of ⁶⁸Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of ⁶⁸Ga are taken into account.

• Biology

  
  Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons David Svilar^1,2, Conchita Vens³, Robert W. Sobol^{1,2,4 1}Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, ²Hillman Cancer Center, University of Pittsburgh Cancer Institute, ³Department of Experimental Therapy, The Netherlands Cancer Institute, ⁴Department of Human Genetics, University of Pittsburgh School of Public Health We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.