Bio-protocol

The endosomal sorting complex required for transport (ESCRT) machinery mediates membrane fission reactions that exhibit a different topology from that observed in clathrin-coated vesicles. In all of the ESCRT-mediated events, the nascent vesicle buds away from the cytosol. However, ESCRT proteins are able to act upon membranes with different geometries. For instance, the formation of multivesicular bodies (MVBs) and the biogenesis of extracellular vesicles both require the participation of the ESCRT-III sub-complex, and they differ in their initial membrane geometry before budding starts: the protein complex acts either from outside the membrane organelle (causing inward budding) or from within (causing outward budding). Several studies have reconstituted the action of the ESCRT-III subunits in supported bilayers and cell-sized vesicles mimicking the geometry occurring during MVBs formation (in-bud), but extracellular vesicle budding (out-bud) mechanisms remain less explored, because of the outstanding difficulties encountered in encapsulation of functional ESCRT-III in vesicles. Here, we provide a different approach that allows the recreation of the out-bud formation, by combining giant unilamellar vesicles as a membrane model and a microinjection system. The vesicles are immobilized prior to injection via weak adhesion to the chamber coverslip, which also ensures preserving the membrane excess area required for budding. After protein injection, vesicles exhibit outward budding. The approach presented in this work can be used in the future to disentangle the mechanisms underlying ESCRT-III-mediated fission, recreating the geometry of extracellular bud production, which remains a challenge. Moreover, the microinjection methodology can be also adapted to interrogate the action of other cytosolic components on the encapsulating membranous organelle.Graphic abstract: Out-bud formation after ESCRT-III protein injection into GUVs

Vasil Georgiev

Yunuen Avalos-Padilla

Xavier Fernàndez-Busquets

Rumiana Dimova

BIO-PROTOCOL

Femtoliter Injection of ESCRT-III Proteins into Adhered Giant Unilamellar Vesicles

飞升体积ESCRT-III蛋白注入贴附的巨型单层囊泡

Gene knock-down in plants is a useful approach to study genotype-phenotype relationships, render disease resistance to crops, and enable efficient biosynthesis of molecules in plants. Small interfering RNA (siRNA)-mediated gene silencing is one of the most common ways to achieve gene knock-down in plants. Traditionally, siRNA is delivered into intact plant cells by coding the siRNA sequences into DNA vectors, which are then delivered through viral and/or bacterial methods. In this protocol, we provide an alternative direct delivery method of siRNA molecules into intact plant cells for efficient transient gene knock-down in model tobacco plant, Nicotiana benthamiana, leaves. Our approach uses one dimensional carbon-based nanomaterials, single-walled carbon nanotubes (SWNTs), to deliver siRNA, and does not rely on viral/bacterial delivery. The distinct advantages of our method are i) there is no need for DNA coding of siRNA sequences, ii) this abiotic method could work in a broader range of plant species than biotic methods, and iii) there are fewer regulatory complications when using abiotic delivery methods, whereby gene silencing is transient without permanent modification of the plant genome. Graphic abstract.

Gozde Demirer

Markita Landry

Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal

Hongzhe Li

Sandro Bräunig

Stefan Scheding

Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing

人骨髓非造血细胞的分离用于单细胞RNA测序

The envelope of Gram-negative bacteria consists of an outer membrane (OM), a peptidoglycan cell wall, and an inner membrane (IM). The OM and IM have different components of proteins and lipids. Separating the IM and OM is a basic biochemical procedure to further study lipids and membrane proteins in different locations. Sucrose gradient ultracentrifugation of lysozyme/EDTA-treated total membrane is the most widely used method to separate the IM and OM of Gram-negative bacteria. However, EDTA is often harmful to protein structure and function. Here, we describe a relatively simple sucrose gradient ultracentrifugation method to separate the IM and OM of Escherichia coli. In this method, the cells are broken by a high-pressure microfluidizer, and the total cell membrane is collected by ultracentrifugation. The IM and OM are then separated on a sucrose gradient. Because EDTA is not used, this method is beneficial for subsequent membrane protein purification and functional study.

Sheng Shu

Wei Mi

Separating Inner and Outer Membranes of Escherichia coli by EDTA-free Sucrose Gradient Centrifugation

Although it is possible to use a tartrate-resistant acid phosphatase (TRAP) stain to assist in identifying osteoclasts, a separate method is needed to determine the bone resorption activity of osteoclasts. Since osteoclasts leave "pits" after bone matrix resorption (Charles et al., 2014), it is possible to stain pits as a method of measuring osteoclast bone resorption activity. The pit assay protocol enables researchers to stain bony slices that were co-cultured with osteoclasts with toluidine blue in order to allow the visualization, capture, and analysis of osteoclast resorptive activity based on the number, size and depth of pits (Zhou et al., 2015). The pit assay protocol is separated into three sequential stages: Preparation of bone slices (1); preparation of osteoclast precursors (Ross et al., 2006; Teitelbaum et al., 2000) (2), and bone resorption pit assay (3).

Alexander Vesprey

Wentian Yang

Pit Assay to Measure the Bone Resorptive Activity of Bone Marrow-derived Osteoclasts

Key features• Standardise the process of building nanobody libraries and finding nanobody binders so that it can be repeated in any lab with reasonable equipment.• Introduce two suites of vectors to functionalise nanobodies for production in either bacterial or mammalian cells.

Lauren E- Eyssen

Siva Ramadurai

Sahar Abdelkarim

Imogen Buckle

Katy Cornish

Hong Lin

A Jones

Gary Stephens

Raymond Owens

From Llama to Nanobody: A Streamlined Workflow for the Generation of Functionalised VHHs

从羊驼到纳米抗体：功能化VHHs的简化工作流程

is an opportunistic pathogen that may cause serious infections in an immunocompromised host.

Pirjo Pärnänen

Timo Sorsa

Taina Tervahartiala

Pirjo Nikula-Ijäs

Preparation and Purification of β-1,3-glucan-Linked &lt;em&gt;Candida glabrata&lt;/em&gt; Cell Wall Proteases by Ion-Exchange Chromatography, Gel Filtration, and MDPF-Gelatin-Zymography Assay

β-1,3-葡聚糖连接的<em>酿酒酵母</em>细胞壁蛋白酶的制备与纯化：离子交换色谱、凝胶过滤和MDPF-明胶-酶谱分析

Proliferating cells need to cope with extensive cytoskeletal and nuclear remodeling as they prepare to divide. These events are tightly regulated by the nuclear translocation of the cyclin B1-CDK1 complex, that is partly dependent on nuclear tension. Standard experimental approaches do not allow the manipulation of forces acting on cells in a time-resolved manner. Here, we describe a protocol that enables dynamic mechanical manipulation of single cells with high spatial and temporal resolution and its application in the context of cell division. In addition, we also outline a method for the manipulation of substrate stiffness using polyacrylamide hydrogels. Finally, we describe a static cell confinement setup, which can be used to study the impact of prolonged mechanical stimulation in populations of cells. Key features • Protocol for microfabrication of confinement devices. • Single-cell dynamic confinement coupled with high-resolution microscopy. • Static cell confinement protocol that can be combined with super-resolution STED microscopy. • Analysis of the mechanical control of mitotic entry in a time-resolved manner.

Margarida Dantas

Débora Vareiro

Jorge G Ferreira

Dissecting the Mechanical Control of Mitotic Entry Using a Cell Confinement Setup

剖析细胞限制装置对有丝分裂进入的力学调控

Zhou Yu

Dongying Wang

Zhiyu Lin

Haixing Li

Protocol to Mine Unknown Flanking DNA Using PER-PCR for Genome Walking

利用PER-PCR挖掘未知侧翼DNA的基因组步移方案

Christopher Groen

Anthony Windebank

Isolation of Intact Mitochondria From &lt;em&gt;Drosophila melanogaster&lt;/em&gt; and Assessment of Mitochondrial Respiratory Capacity Using Seahorse Analyzer

从果蝇中分离完整线粒体并使用Seahorse分析仪评估线粒体呼吸能力

Abbreviation	Bio. Protoc.
Journal Impact	0.95
Quartiles(Global)	BIOLOGY(Q3)

ISSN	2331-8325
h-index	18

Publication Information	Publisher: Bio-protocol LLC，Publishing cycle: ，Journal Type: journal，Open Access Journals: Yes
Basic data	Year of publication: 2018，Proportion of original research papers: 100.00%，Self Citation Rate:， Gold OA Rate: 14.87%
Journal Fees	$1200Details
Average review cycle	12 weeks

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