| Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue
|
|
Author:
Date:
2017-12-20
[Abstract] Skeletal muscle provides the contractile force necessary for movement, swallowing, and breathing and, consequently, is necessary for survival. Skeletal muscle cells are unique in that they are extremely large cells containing thousands of nuclei. These nuclei must all work in concert to maintain skeletal muscle function and thereby maintain life. The nucleus is a major site of signaling integration and gene expression regulation. However, examining nuclear processes in skeletal muscle can be difficult because myonuclei are challenging to isolate. We optimized a protocol to purify myonuclei from whole muscle tissue using ultracentrifugation over a discontinuous sucrose gradient to separate the nuclear fraction. We used these purified nuclei for downstream applications including flow ...
[摘要] 骨骼肌提供运动，吞咽和呼吸所需的收缩力，因此是生存所必需的。骨骼肌细胞是独特的，因为它们是含有数千个核的极大细胞。这些细胞核必须协调一致才能维持骨骼肌功能，从而维持生命。细胞核是信号整合和基因表达调控的主要部位。然而，检查骨骼肌中的核过程可能是困难的，因为肌核难以分离。我们优化了一个协议，从整个肌肉组织使用超速离心蔗糖梯度来分离核分数的纯化肌细胞。我们将这些纯化的细胞核用于下游应用，包括流式细胞术和质谱。我们使用这种方法来比较年轻和老鼠后肢肌肉的肌细胞蛋白质组（Cutler等人，2017）。这个协议可能适用于分离myonuclei的各种下游分析，如流式细胞仪，显微镜，蛋白质印迹和蛋白质组学。
【背景】为了生存，必须保持适当的骨骼肌功能。这种维持的一个组成部分是根据细胞需要和环境线索调整基因表达。调节基因表达的核过程是调节细胞组成和行为的关键组成部分。然而，由于四个技术限制，涉及这些过程的肌细胞蛋白难以研究。首先，骨骼肌是致密的，紧密地填满了组织中超过60％的蛋白质的收缩性蛋白质（Deshmukh等人，2015; Cutler等人）。 ...
|
|
|
| Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs
|
|
Author:
Date:
2015-06-05
[Abstract] Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding the association between asymptomatic replication of CMV (and other herpesvirus, including EBV) and this HIV reservoir (Gianella et al., 2014). This protocol was designed to simultaneously co-extract DNA and RNA from the same peripheral blood mononuclear cell (PBMC) aliquot and quantify HIV, CMV and EBV DNA, as well as HIV RNA using droplet digital PCR (ddPCR).
For collection and processing of male genital secretions and quantification of HIV RNA and DNA from seven human herpesviruses from seminal plasma, refer to ...
[摘要] 几乎所有感染人免疫缺陷病毒（HIV）的个体也被巨细胞病毒（CMV）和EB病毒（EBV）感染。 我们的研究的目的包括表征和测量潜伏的HIV库并且理解CMV（和其他疱疹病毒，包括EBV）的无症状复制与该HIV库之间的关联（Gianella等人，2014） 。 该方案设计为从相同的外周血单核细胞（PBMC）等分试样同时共提取DNA和RNA，并使用液滴数字PCR（ddPCR）定量HIV，CMV和EBV DNA以及HIV RNA。
关于男性生殖器分泌物的收集和处理以及来自七种人疱疹病毒的HIV RNA和DNA的定量，参见方案"Vacs RNA和人疱疹病毒DNA在Seminal血浆中的定量（Vargas-Meneses等人，2015）。
|
|
|
| Transwell Co-culture of Bone Marrow Macrophages with Tumor Cells
|
|
Author:
Date:
2014-05-05
[Abstract] Bone is a primary site of metastasis from prostate and breast cancers. Bone marrow macrophages (BMMs) are mediators of inflammatory processes and are thought to promote tumor growth in the skeletal sites. In order to elucidate how their interactions with tumor cells impact aggressiveness of metastatic tumors in bone in vitro methods are required. By employing a system in which BMMs and tumor cells are grown separately, yet share the media and exchange soluble factors, contribution of each cell type in the context of BMM-tumor cell relationship in the bone marrow can be investigated. Additional advantages of this system include the ability to study: 1) phenotypic changes in BMMs and tumor cells upon co-culture; 2) cell-specific modulation of protein and gene expression; and 3) ...
[摘要] 骨是来自前列腺和乳腺癌的原发转移部位。骨髓巨噬细胞（BMM）是炎症过程的介质，并且被认为促进骨骼部位中的肿瘤生长。为了阐明它们与肿瘤细胞的相互作用如何影响骨髓中转移性肿瘤的侵袭性，需要体外方法。通过采用其中BMM和肿瘤细胞分开生长，但共享培养基和交换可溶性因子的系统，可以研究在骨髓中BMM-肿瘤细胞关系的上下文中每种细胞类型的贡献。该系统的另外的优点包括研究以下的能力：1）共培养时BMM和肿瘤细胞中的表型改变; 2）蛋白质和基因表达的细胞特异性调节;和3）对增殖和细胞存活的影响。值得注意的是，该transwell共培养系统不限于BMM和肿瘤细胞，并且可以容易地修饰以包括骨髓微环境的其他组分（例如，脂肪细胞，基质细胞，成骨细胞）。
|
|
|