Table of contents

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Interferometric scattering microscopy and its combination with single-molecule fluorescence imaging pp617 - 633

Jaime Ortega Arroyo, Daniel Cole & Philipp Kukura


Interferometric scattering microscopy (iSCAT) enables ultrasensitive label-free imaging and high-speed single-particle tracking. This protocol describes how to construct an iSCAT microscope with single-molecule TIRF capabilities.

Assembly and operation of the autopatcher for automated intracellular neural recording in vivo pp634 - 654

Suhasa B Kodandaramaiah, Gregory L Holst, Ian R Wickersham, Annabelle C Singer, Giovanni Talei Franzesi, Michael L McKinnon, Craig R Forest & Edward S Boyden


This protocol describes how to set up and use the autopatcher, a robot that automatically obtains intracellular neural recordings from intact mammalian mouse brains.

Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry pp655 - 663

Lanzhou Jiang, Rochelle Tixeira, Sarah Caruso, Georgia K Atkin-Smith, Amy A Baxter, Stephanie Paone, Mark D Hulett & Ivan K H Poon


Annexin A5 and TO-PRO-3 (a nucleic acid–binding dye that stains early apoptotic and necrotic cells differentially) are used to distinguish six types of particles in a sample, including apoptotic bodies and cells at various stages of cell death.

Using Raman spectroscopy to characterize biological materials pp664 - 687

Holly J Butler, Lorna Ashton, Benjamin Bird, Gianfelice Cinque, Kelly Curtis, Jennifer Dorney, Karen Esmonde-White, Nigel J Fullwood, Benjamin Gardner, Pierre L Martin-Hirsch, Michael J Walsh, Martin R McAinsh, Nicholas Stone & Francis L Martin


Raman microspectroscopy is useful for the analysis of biological samples, because chemical and structural information can be obtained without using labels. This protocol brings together practical guidelines from expert research groups.

Near-IR photoactivation using mesoporous silica–coated NaYF4:Yb,Er/Tm upconversion nanoparticles pp688 - 713

Muthu Kumara Gnanasammandhan, Niagara Muhammad Idris, Akshaya Bansal, Kai Huang & Yong Zhang


Upconversion nanoparticles (UCNs) have the extraordinary ability to emit light with UV-visible wavelengths on illumination in the near IR region. This protocol describes the preparation of UCNs with potential therapeutic applications.

Microfluidic differential immunocapture biochip for specific leukocyte counting pp714 - 726

Umer Hassan, Nicholas N Watkins, Bobby Reddy Jr, Gregory Damhorst & Rashid Bashir


This protocol from Hassan et al. describes a microfluidic chip that uses an immunocapture chamber to count CD4 and CD8 cells in whole blood for HIV/AIDS diagnostics. The chip can be adapted for different cell types and research applications.

Functionalization, preparation and use of cell-laden gelatin methacryloyl–based hydrogels as modular tissue culture platforms pp727 - 746

Daniela Loessner, Christoph Meinert, Elke Kaemmerer, Laure C Martine, Kan Yue, Peter A Levett, Travis J Klein, Ferry P W Melchels, Ali Khademhosseini & Dietmar W Hutmacher


This protocol describes how to make semisynthetic gelatin methacryloyl (GelMA)-based hydrogels for use in 3D cell culture models for cancer research, stem cell research and tissue engineering.

A LC-MS–based workflow for measurement of branched fatty acid esters of hydroxy fatty acids pp747 - 763

Tejia Zhang, Shili Chen, Ismail Syed, Marcus Ståhlman, Matthew J Kolar, Edwin A Homan, Qian Chu, Ulf Smith, Jan Borén, Barbara B Kahn & Alan Saghatelian


Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biological lipids. This protocol describes their extraction from serum and tissue samples, followed by enrichment and analysis by LC-MS.

Automated screening for small organic ligands using DNA-encoded chemical libraries pp764 - 780

Willy Decurtins, Moreno Wichert, Raphael M Franzini, Fabian Buller, Michael A Stravs, Yixin Zhang, Dario Neri & Jörg Scheuermann


This protocol describes an approach for screening DNA-encoded chemical libraries (DECLs) to identify molecules that bind to proteins of interest. After isolating binding library members, DNA barcodes are amplified and identified by high-throughput sequencing.

Design, synthesis and evaluation of molecularly targeted hypoxia-activated prodrugs pp781 - 794

Liam J O'Connor, Cindy Cazares-Körner, Jaideep Saha, Charles N G Evans, Michael R L Stratford, Ester M Hammond & Stuart J Conway


Many solid tumors contain an aggressive hypoxic region that is difficult to treat. This protocol describes how to prepare bioreductive prodrugs that are biologically inactive until they are converted to an active drug by enzymatic reduction in hypoxia.

Label-free quantification in ion mobility–enhanced data-independent acquisition proteomics pp795 - 812

Ute Distler, Jörg Kuharev, Pedro Navarro & Stefan Tenzer


This protocol describes a data-independent acquisition workflow for label-free quantitative proteomics that integrates ion mobility separation and applies drift time–specific collision energies to improve precursor fragmentation efficiency.

Genome-wide profiling of RNA polymerase transcription at nucleotide resolution in human cells with native elongating transcript sequencing pp813 - 833

Andreas Mayer & L Stirling Churchman


Human NET-seq enables DNA strand–specific mapping of RNA polymerase (RNAP) activity at single-nucleotide resolution. A cell fractionation approach is used to isolate transcribing RNAP and associated RNAs, avoiding immunoprecipitation or RNA labeling.


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