Novel application of flow cytometry: Determination of muscle fiber types and protein levels in whole murine skeletal muscles and heart
Novel application of flow cytometry: Determination of muscle fiber types and protein levels in whole murine skeletal muscles and heart
复制标题
DOI：
10.1002/cm.20239
复制
发表时间：
2007-12-01
影响因子：
--
通讯作者：
Laing, Nigel G.
中科院分区：
文献类型：
--
作者：
Jackaman, Connie;Nowak, Kristen J.;Laing, Nigel G.
测量肌肉样品中蛋白质的传统方法，如免疫组织化学和western blot分析，可能是耗时的，劳动密集型的，并受到采样误差的影响。我们已经开发了流式细胞术技术来检测整个小鼠心脏和骨骼肌中的蛋白质。对雄性C57BL/6J、BALB/c、CBA和mdx小鼠的股四头肌和比目鱼肌进行流式细胞术和免疫组化。单染流式细胞术检测肌动蛋白、肌球蛋白、原肌球蛋白和α -肌动蛋白。这与使用相同抗体的免疫组织化学反应有关。骨骼肌肌动蛋白和不同肌球蛋白的双标记流式细胞术可测定肌纤维类型。这与I、IIA和IIB肌球蛋白的免疫组化结果相似。用心脏和骨骼肌肌动蛋白抗体双标记的C57BL/6J和BALB/c小鼠心脏样本的流式细胞术显示，已知BALB/c心脏中骨骼肌动蛋白增加。在C57BL/6J小鼠中可检测到α -肌糖聚糖和肌营养不良蛋白，而在mdx小鼠中则减少或不存在。由于能够同时用多种抗体标记整个肌肉样本，流式细胞术在某些应用中可能比传统方法更有优势，包括评估肌肉疾病潜在疗法的疗效。
Conventional methods for measuring proteins within muscle samples such as immunohistochemistry and western blot analysis can be time consuming, labor intensive and subject to sampling errors. We have developed flow cytometry techniques to detect proteins in whole murine heart and skeletal muscle. Flow cytometry and immunohistochemistry were performed on quadriceps and soleus muscles from male C57BL/6J, BALB/c, CBA and mdx mice. Proteins including actins, myosins, tropomyosin and alpha-actinin were detected via single staining flow cytometric analysis. This correlated with immunohistochemistry using the same antibodies. Muscle fiber types could be determined by dual labeled flow cytometry for skeletal muscle actin and different myosins. This showed similar results to immunohistochemistry for I, IIA and IIB myosins. Flow cytometry of heart samples from C57BL/6J and BALB/c mice dual labeled with cardiac and skeletal muscle actin antibodies demonstrated the known increase in skeletal actin protein in BALB/c hearts. The membrane-associated proteins alpha-sarcoglycan and dystrophin could be detected in C57BL/6J mice, but were decreased or absent in mdx mice. With the ability to label whole muscle samples simultaneously with multiple antibodies, flow cytometry may have advantages over conventional methods for certain applications, including assessing the efficacy of potential therapies for muscle diseases.