Author:
Date:
2018-02-05
[Abstract] The Smurf Assay (SA) was initially developed in the model organism Drosophila melanogaster where a dramatic increase of intestinal permeability has been shown to occur during aging (Rera et al., 2011). We have since validated the protocol in multiple other model organisms (Dambroise et al., 2016) and have utilized the assay to further our understanding of aging (Tricoire and Rera, 2015; Rera et al., 2018). The SA has now also been used by other labs to assess intestinal barrier permeability (Clark et al., 2015; Katzenberger et al., 2015; Barekat et al., 2016; Chakrabarti et al., 2016; Gelino et al., 2016). The SA in itself is simple; however, numerous small details can have a considerable impact on its ...
[摘要] Smurf分析(SA)最初是在模型生物黑腹果蝇中发现的,其中在老化过程中肠道通透性显着增加(Rera等人,2011年)。 我们已经验证了多种其他模式生物体的协议(Dambroise等人,2016),并利用该分析来进一步了解老化(Tricoire和Rera,2015; Rera等人 。,2018年)。 SA现在也被其他实验室用于评估肠道屏障通透性(Clark等人,2015; Katzenberger等人,2015; Barekat等人 。2016; Chakrabarti等人,2016; Gelino等人,2016)。 SA本身很简单, 然而,许多小细节可能会对其实验的有效性和随后的解释产生相当大的影响。 在这里,我们提供SA技术的详细更新,并解释如何捕捉Smurf,同时避免最常见的实验谬误。
【背景】Smurf分析(SA)基于(Wong等人,2009)中描述的果蝇饲养分析。该测定法通过摄入未被消化道吸收的蓝色染料来评估食物摄入量,从而允许直接定量。对于SA来说,这个特定的蓝色染料不能通过一个完整的肠屏障,因为它的读数是全身着色(这里是蓝色)。这种性质允许直接评估肠道通透性,已经显示其随着年龄的增长而增加(Rera等人,2011和2012)。
正如最近在Rera等人(2018)中所讨论的那样,Smurf分析不仅是评估体内肠渗透性的简单方法,而且也是评估广泛生物体中个体的生理年龄。因此,它允许以新颖的方法来研究在老化个体中发生的各种事件(Tricoire和Rera,2015; ...
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Author:
Date:
2017-04-20
[Abstract] Cellular barcoding enables the dissection of clonal dynamics in heterogeneous cell populations through single cell lineage tracing. The labeling of hematopoietic stem and progenitor cells (HSPCs) with unique and heritable DNA barcodes, makes it possible to resolve donor cell heterogeneity in terms of differentiation potential and lineage bias at the single cell level, through subsequent transplantation and high-throughput sequencing. Furthermore, cellular barcoding allows for bona fide hematopoietic stem cells (HSCs) to be defined based on functional rather than immunophenotypic parameters.
This protocol describes the work flow of lentiviral cellular barcoding, tracking 14.5 days post coitum (d.p.c.) fetal liver (FL) Lineage-Sca+cKit+ (LSK) HSPCs following ...
[摘要] 细胞条形码可以通过单细胞谱系追踪来分离异种细胞群体中的克隆动力学。通过独特和可遗传的DNA条形码对造血干细胞和祖细胞(HSPC)进行标记,可以通过随后的移植和高通量测序,在单细胞水平上分化供体细胞异质性,分化潜力和谱系偏倚。此外,细胞条形码可以根据功能而不是免疫表型参数定义真正的造血干细胞(HSC)。  该协议描述了慢病毒细胞条形码的工作流程,追踪1450天后(dpc)胎肝(FL)Lineage-Sca+ cKit + (LSK)HSPC经过长期重建(图1)(Kristiansen等人,2016),但可以适应于选择的细胞类型或时间框架。
图1.实验工作流程摘要(Naik 等人,2013)
最初建立了细胞条形码技术来解决在体内移植造血细胞后的单细胞动力学,并且近年来在移植中对血细胞群体中功能异质性的认识有显着贡献(Schepers ,2008; Gerrits等人,2010; Lu等人,2011; Naik等人 ,2013; Verovskaya等人,2013; ...
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