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Trypsin (0.25%), phenol red


Company: Thermo Fisher Scientific
Catalog#: 15050065
Other protocol()

In vitro Assays for the Detection of Calreticulin Exposure, ATP and HMGB1 Release upon Cell Death
[Abstract]  Accumulating evidence is revealing the essential role of immune system in cancer treatment. Certain chemotherapeutic drugs can potently induce the release of ‘cell death associated molecular patterns’ (CDAMPs), which accompanies cancer cell demise. CDAMPs can engage corresponding receptors on immune cells and stimulate immune responses to achieve long-term tumor control (Ma et al., 2013; Ma et al., 2014; Yang et al., 2015). Among reported CDAMPs, calreticulin (CALR), ATP and HMGB1 are well known for their immune-stimulatory effect. Here we describe the assays that we applied to measure cell death and these CDAMPs. Briefly, cell death can be analyzed by co-staining of 4’,6-diamidino-2-phenylindole (DAPI) with 3,3’-Dihexyloxacarbocyanine Iodide [DiOC6(3)] or ... [摘要]  积累的证据表明免疫系统在癌症治疗中的重要作用。某些化学治疗药物可以有效诱导伴随癌细胞死亡的“细胞死亡相关分子模式”(CDAMPs)的释放。 CDAMP可以与免疫细胞上的相应受体结合并刺激免疫应答以实现长期的肿瘤控制(Ma等,2013; Ma等人,2014; Yang& em>等,,2015)。在所报道的CDAMP中,钙网蛋白(CALR),ATP和HMGB1是众所周知的免疫刺激作用。在这里我们描述我们应用于测量细胞死亡和这些CDAMPs的测定。简单地说,细胞死亡可以通过4',6-二脒基-2-苯基吲哚(DAPI)与3,3'-二氧基氧羰基花青氨基碘[DiOC6(3)]或Annexin V的共染色来分析。通过流式细胞术检测。 ATP和HMGB1释放可以通过发光测定和ELISA测定分别进行定量。

背景 乳酸脱氢酶测定和台盼蓝染色是检测细胞死亡的传统方法。我们在这里描述两种可行和经济的解决方案,以通过流式细胞术(FCM)检测凋亡和坏死细胞死亡。 DAPI标记具有破坏完整性(坏死)的细胞,而膜联蛋白V结合磷脂酰丝氨酸(其在凋亡后被外化)。 DiOC6(3)摄取表明线粒体跨膜电位(MTP),MTP的崩解显示凋亡。 DAPI不需要补偿藻红蛋白(PE,其与膜联蛋白V蛋白结合)或DiOC6(3),因此在这些测定中显示优于碘化丙啶(PI)的优点。 ...

Cell Culture Transfection for Production and Purification of Wnt Ligands
[Abstract]  Wnt ligand proteins are extremely difficult to purify and enrich in vitro. This protocol uses Wnt11r protein as an example to illustrate how to use 293T cells to produce secreted Wnt11r and collect it in vitro for further biochemical experiments. [摘要]  Wnt配体蛋白在体外非常难以纯化和富集。 该协议使用Wnt11r蛋白作为例子说明如何使用293T细胞产生分泌的Wnt11r,并收集在体外进行进一步的生化实验。