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Nature Protocols Contents: Volume 13 Number 10

Nature Protocols


October 2018 Volume 13, Issue 10


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Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry    pp2121 - 2148
Guojun Han, Matthew H. Spitzer, Sean C. Bendall, Wendy J. Fantl & Garry P. Nolan

The immunoassay multiplexing capacity of single-cell mass cytometry relies on monoclonal antibodies labeled with stable heavy-metal isotopes. Nolan et al. describe procedures for conjugating monoclonal IgG antibodies with 48 heavy-metal isotopes.

Acute and rapid degradation of endogenous proteins by Trim-Away    pp2149 - 2175
Dean Clift, Chun So, William A. McEwan, Leo C. James & Melina Schuh

This protocol describes Trim-Away, an approach for rapid protein depletion in different cell types. TRIM21–mediated proteasomal degradation is induced by microinjection or electroporation of an antibody into the protein of interest.

Mixed-species RNA-seq for elucidation of non-cell-autonomous control of gene transcription    pp2176 - 2199
Jing Qiu, Owen Dando, Paul S. Baxter, Philip Hasel, Samuel Heron et al.

This protocol describes the co-culture of cells from multiple species and, after RNA-seq, the separation of reads by species via the Sargasso bioinformatics pipeline to elucidate the effects of one cell type on the transcriptome of the others.

Mapping heterogeneity of cellular mechanics by multi-harmonic atomic force microscopy    pp2200 - 2216
Yuri M. Efremov, Alexander X. Cartagena-Rivera, Ahmad I. M. Athamneh, Daniel M. Suter & Arvind Raman

This protocol describes a dynamic atomic force microscopy (dAFM) approach for high-speed and high-resolution mapping of the viscoelastic properties of live cells. The procedure describes sample preparation, AFM calibration, and data analysis.

Genetic lineage tracing of resident stem cells by DeaLT    pp2217 - 2246
Lingjuan He, Yan Li, Xiuzhen Huang, Yi Li, Wenjuan Pu et al.

In this protocol, the authors explain a new, more precise genetic-lineage-tracing system based on a dual-recombinase strategy. DeaLT enables specific fate mapping of resident stem cells by using both the Cre–loxP and Dre–rox systems.

An experimental murine model to study periodontitis    pp2247 - 2267
Julie Marchesan, Mustafa S. Girnary, Li Jing, Michael Zhe Miao, Shaoping Zhang et al.

In this protocol, a ligature is placed between mouse teeth. This induces gingival tissue inflammation and alveolar bone loss, resulting in a mouse model of periodontitis.

Single-mRNA detection in living S. cerevisiae using a re-engineered MS2 system    pp2268 - 2296
Evelina Tutucci, Maria Vera & Robert H. Singer

This protocol describes how to use a re-engineered MS2 system to image single mRNAs in Saccharomyces cerevisiae. The procedure describes tagging of endogenous genes with MBSV6, two-color smFISH, and how to quantify single mRNAs by live cell imaging.

In vivo pulse labeling of isochronic cohorts of cells in the central nervous system using FlashTag    pp2297 - 2311
Subashika Govindan, Polina Oberst & Denis Jabaudon

This protocol describes an approach for pulse labeling of ventricular zone progenitors and their neuronal progeny in the developing brain. Labeled cells can be isolated and highlighted by FACS or immunohistochemistry.

Design and operation of reconfigurable two-dimensional DNA molecular arrays    pp2312 - 2329
Dongfang Wang, Jie Song, Pengfei Wang, Victor Pan, Yingwei Zhang et al.

This protocol describes how to design and assemble two-dimensional reconfigurable DNA arrays that can be used for long-range information relay. The procedure describes the design principles and AFM- and TEM-based imaging of the structures.

Direct fluorine-18 labeling of heat-sensitive biomolecules for positron emission tomography imaging using the Al18F-RESCA method    pp2330 - 2347
Frederik Cleeren, Joan Lecina, Jessica Bridoux, Nick Devoogdt, Térence Tshibangu et al.

This protocol describes a method for direct fluorine-18 labeling of heat-sensitive proteins for PET imaging. After conjugation to RESCA chelators, proteins of interest can be radiolabeled with Al18F at room temperature in an aqueous medium.

Synthesis of an ultrasensitive BODIPY-derived fluorescent probe for detecting HOCl in live cells    pp2348 - 2361
Hao Zhu, Zhen Zhang, Saran Long, Jianjun Du, Jiangli Fan et al.

This protocol describes the one-pot chemical synthesis and applications of BClO, an ultrasensitive fluorescent probe for detecting hypochlorous acid (HOCl) in live cells.

Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors    pp2362 - 2386
Yejun Zou, Aoxue Wang, Mei Shi, Xianjun Chen, Renmei Liu et al.

This protocol describes how to combine up to four genetically encoded fluorescent sensors to image redox landscapes. The procedure describes applications in live imaging and flow cytometry of cultured cells, and in vivo imaging in zebrafish larvae.

Use of the iNo score to discriminate normal from altered nucleolar morphology, with applications in basic cell biology and potential in human disease diagnostics    pp2387 - 2406
Vassiliki Stamatopoulou, Pascaline Parisot, Christophe De Vleeschouwer & Denis L. J. Lafontaine

This protocol qualitatively and quantitatively assesses the involvement of factors in the nucleolar structure. After siRNA-mediated depletion of factors of interest, dedicated software is used to analyze images of nucleoli to determine an index of nucleolar disruption, the iNo score.

Small-seq for single-cell small-RNA sequencing    pp2407 - 2424
Michael Hagemann-Jensen, Ilgar Abdullayev, Rickard Sandberg & Omid R Faridani

Faridani and colleagues describe Small-seq, a protocol for generating sequencing libraries of small RNAs from single cells.

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