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 ...
[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)。 ...
|
|
Stable Isotope Resolved Metabolomics Studies in ex vivo TIssue Slices
|
Author:
Date:
2016-02-05
[Abstract] An important component of this methodology is to assess the role of the tumor microenvironment on tumor growth and survival. To tackle this problem, we have adapted the original approach of Warburg (Warburg, 1923), by combining thin tissue slices with Stable Isotope Resolved Metabolomics (SIRM) to determine detailed metabolic activity of human tissues. SIRM enables the tracing of metabolic transformations of source molecules such as glucose or glutamine over defined time periods, and is a requirement for detailed pathway tracing and flux analysis. In our approach, we maintain freshly resected ...
[摘要] 这种方法的一个重要组成部分是评估肿瘤微环境对肿瘤生长和存活的作用。为了解决这个问题,我们采用Warburg(Warburg,1923)的原始方法,通过将薄组织切片与稳定同位素解析代谢组学(SIRM)组合来确定人体组织的详细代谢活性。 SIRM使得能够在定义的时间段内跟踪诸如葡萄糖或谷氨酰胺的来源分子的代谢转化,并且是详细的途径追踪和通量分析的要求。在我们的方法中,我们保持在细胞培养基中新鲜切除的组织切片(来自同一受试者的相同器官的癌性和非癌性),并用适当的稳定的富含同位素的营养物,例如葡萄糖或13 C 15葡萄糖或13 C 15葡萄糖或13 C 15葡萄糖, - 谷氨酰胺。这些切片可存活至少24小时,并使得有可能消除对靶组织代谢的系统影响,同时保持原始的3D细胞结构。因此,它是用于评估治疗剂对靶组织代谢的影响及其对个体患者的治疗功效的极好的临床前平台(Xie等人,2014; Sellers等人,/em>,2015)。
|
|
Immune Cell Isolation from Mouse Femur Bone Marrow
|
Author:
Date:
2015-10-20
[Abstract] The bone marrow is the site of hematopoiesis and contains mixed population of blood cells including erythrocytes, granulocytes, monocytes, dendritic cells, lymphocytes and hematopoietic stem cells. The following protocol provides a simple and fast method for isolation of bone marrow immune cells (no erythrocytes) from mouse femurs with a yield of approximate 8 x 107 cells in 5 ml culture media (1.6 x 104 cells/µl). Further isolation or flow cytometric analysis might be required for study of specific immune cell types.
[摘要] 骨髓是造血的部位,包含红细胞,粒细胞,单核细胞,树突状细胞,淋巴细胞和造血干细胞等混合血细胞。 以下方案提供了一种简单而快速的方法,用于在5ml培养基(1.6×10 4个细胞/μl)中分离来自小鼠股骨的骨髓免疫细胞(无红细胞),产量约为8×10 7个细胞。 研究特异性免疫细胞类型可能需要进一步的分离或流式细胞分析。
|
|