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The Netherlands Cancer Institute

4 ARTICLES PUBLISHED IN JoVE

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Biology

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
David Svilar 1,2, Conchita Vens 3, Robert W. Sobol 1,2,4
1Department 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.

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Biochemistry

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery
Misbha Ud Din Ahmad 1, Alexander Fish 1, Jeroen Molenaar 1, Sridhar Sreeramulu 2, Christian Richter 2, Nadide Altincekic 2, Harald Schwalbe 2, Hans Wienk 1, Anastassis Perrakis 1
1Oncode 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.

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Biology

Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling
Dimitrios Laskaris *1,2, Maria Azkanaz *1,2, Mijke A. de Vreij-Kruidenier 3, Doreen van Rijswoud-Ram 3, Hendrik A. Messal 1,2, Jacco van Rheenen 1,2
1Department 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.

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Developmental Biology

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing
Naomi J. M. Kakiailatu 1, Laura E. Kuil 1,2, Eric Bindels 3, Joke T. M. Zink 3, Michael Vermeulen 3, Veerle Melotte 4, Maria M. Alves 1
1Department of Clinical Genetics, Erasmus University Medical Center - Sophia Children's Hospital, 2Division of Psychosocial Research and Epidemiology, the Netherlands Cancer Institute, 3Department of Hematology, Erasmus Medical Center, 4Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

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