在肾小管上皮细胞胞浆钙的测量流式细胞仪
Summary
钙是参与许多生理学和病理生理学的信号通路。活细胞成像，需要专门的设备和非常耗时。使用流式细胞仪来确定在纳入悬浮贴壁的上皮细胞内钙的相对变化快速，简单的方法进行了优化。
Abstract
A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.
Introduction
钙是负责细胞的生理学和病理生理学信号1的发送的重要的第二信使。细胞内钙的浓度保持在较低水平，通过钙泵和钙交换体的活性。肌浆/内质网钙ATP酶（SERCA）重新充质网（ER）钙库作为“泵泄漏”系统而质膜钙ATP酶（PMCA）挤出钙向细胞外隔室的一部分，无论是在ATP依赖的方式2。生理信使，例如激素或神经递质，通过G-蛋白偶联受体，激活磷脂酶C，其水解磷脂酰肌醇4,5-二磷酸（PIP2）在质膜中，以产生二酰基甘油和三磷酸肌醇（IP3）3的发送它们的信号。而二酰基甘油保留在质膜上，IP3扩散进入细胞溶胶和结合的IP3受体秒（IP3Rs），它们的配体激活的钙通道，在ER膜和钙被发现从ER腔商店最终于增加细胞内钙浓度的4释放。对于钙到达胞质溶胶的替代路线是通过钙离子通道和热交换器中存在的质膜。这两个车厢之间的串扰进行了描述：钙诱导钙释放（CICR），其中的细胞外钙离子诱导的ER店5钙释放和存储操作的钙进入（SOCE），其中排空ER店是由STIM感应到，并导致开放Orai的钙通道在细胞膜，促进ER商店6的再填充。
在病理情况下，细胞的钙反应是放松管制和细胞内钙增加的情况下的生理刺激1。钙反应可能会影响在许多方面：长期钙信号，延迟钙去除胞浆钙商店或钙局部变化的枯竭。此外，线粒体承担了中心作用在缓冲和释放钙7。的延长和/或超大的细胞内钙的增加导致细胞死亡。事实上，钙是往往不是参与细胞病理生理反应，是在疾病，如神经变性疾病，心脏疾病，癌症，骨疾病，肾疾病，这主要与细胞死亡有关的各种各样的键事件和丢失或改动的器官或组织功能8-10。此外，钙信号的扰动已与细胞适应和改变的细胞功能和响应。
传统上，钙信号被测量与被加载到细胞中的细胞可渗透的乙酰氧基甲基（AM）酯形式的带负电的荧光钙指示剂。一旦通过细胞酯酶切割上课时，荧光指示器保持在胞内区室中，增加的荧光强度时，钙绑定。最知名的和使用的钙指标是比例的Fura-2罗杰钱永健和他的同事开发出11。用不同的亲和力对钙的钙指标允许不同的钙池进行监控。检测方法包括活细胞成像，荧光酶标仪，流式细胞仪。钙信号的相对快速动力学（通常几秒钟至几分钟内），使活细胞成像获取关于钙信号的特性的最信息的最佳方法。除了 钙信号（在几毫秒内）的快速动力学，活细胞成像是单细胞12内研究钙信号的细胞区室化的较好方法。绝对钙浓度可通过测定钙的ind的最小和最大荧光来计算icator通过添加钙离子螯合剂和透化的细胞，分别如由Grynkiewicz 等人 11。
首先被开发于1980年代在免疫细胞中的使用流式细胞仪来测量钙信号，其中的机会，以测量多个参数，例如膜完整性和细胞群的分离，并且细胞在悬浮液中的要求并结合单波长的发展钙指标方面流式细胞仪的理想和convenientdetection方法13-15。在贴壁细胞，活细胞成像提供钙信号传导，其中最重要的动力学的信息最多，但需要复杂的设置，其包括荧光显微镜，灌注系统，维护蜂窝环境，如温度，并专门显微镜软件，用于获取和分析数据。替代的方法如荧光酶标仪或药理学通过使用钙离子螯合剂ogical手段所无法比拟的获得信息的条款。流式细胞术变得越来越广泛地用于监测在贴壁细胞的细胞内钙虽然在大多数的研究中，测量只进行终点。在免疫的B细胞16使用由Gergely的等最初开发的方法。通过流式细胞仪具有实时动力学和监测从一个样品群体的单个细胞的荧光强度的变化在细胞内游离钙离子浓度已成功地在肿瘤上皮细胞中测得的和癌症干细胞杂种17。该方法还适合于贴壁的上皮细胞，这是难以负荷与钙指标18使用。
这里，使用从大鼠肾近端小管（WKPT-0293 Cl.2）19的S1片段衍生的永生化粘附上皮细胞系，我们描述了一种优化的简化米ethod用于通过流式细胞术改变细胞内钙浓度的测定。因为许多上皮细胞具有许多的阴离子化合物的有机转运，细胞装载钙指示剂的可以被证明是一个挑战。为了防止钙的指标，丙磺舒，有机阴离子转运原型抑制剂和最初开发来降低青霉素20的肾脏排泄外排，是在培养液同时使用。将细胞维持在单层钙指示剂装载，带入悬浮液和钙信号可化合物加成后，立即检测到。
Protocol
Representative Results
Discussion
钙是在充当第二信使信号传导分子，也可以作为内质网和线粒体之间进行通信的装置，多种细胞过程的关键事件。来测算胞浆游离钙离子浓度的能力是可以应用于细胞生物学的各个领域一个非常有用的技术。存在各种方法来检测细胞内钙信号。经典地，从一个比例钙指示剂，如呋喃-2信号，在使用荧光成像的设置和绝对钙浓度的活细胞监测可衍生自确定的最小和最大荧光强度。但是，这种技术需?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
研究实验室是由弗里茨 – 本德基金会，慕尼黑，德国（以TD）和威腾/黑尔德克内部研究资助大学（以W.-KL）资助。我们要感谢博士教授弗兰克博士Thévenod（研究所生理学，病理生理学和毒理学，威腾大学/黑尔德克）的有益的建议。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Fluo-3, AM | Invitrogen | F-1241 | Cell permeable |
| Probenecid | Sigma-Aldrich | P-8761 | Water insoluble, dissolve in 1 N NaOH |
| 0.05% Trypsin-EDTA (1X) | Invitrogen | 25300-062 | |
| Ionomycin | Sigma-Aldrich | I9657 | |
| Thapsigargin | Tocris Bioscience | 1138 | |
| Tunicamycin | Sigma-Aldrich | T7765 | |
| FACSCalibur Flow Cytometer + CELLQuest software | Becton Dickinson | ||
| Windows Multiple Document Interface for Flow Cytometry (WinMDI) | Joe Trotter, The Scripps Institute | Please note that this is an older 16-bit application that reads FCS 2.0 compliant files but does not recognize FCS 3.0 digital data. | |
| WKPT-0293 Cl.2 rat kidney proximal tubule cell line | Made available by Dr. Ulrich Hopfer (Department of Physiology & Biophysics, Case Western Reserve University, Cleveland, OH) |
References
- Berridge, M. J., Lipp, P., Bootman, M. D. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol. 1, 11-21 (2000).
- Brini, M., Carafoli, E. Calcium pumps in health and disease. Physiol Rev. 89, 1341-1378 (2009).
- Berridge, M. J. Inositol trisphosphate and calcium signalling. Nature. 361, 315-325 (1993).
- Putney, J. W. Formation and actions of calcium-mobilizing messenger, inositol 1,4,5-trisphosphate. Am J Physiol. 252, 149-157 (1987).
- Putney, J. W., Ribeiro, C. M. Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores. Cell Mol Life Sci. 57, 1272-1286 (2000).
- Soboloff, J., Rothberg, B. S., Madesh, M., Gill, D. L. STIM proteins: dynamic calcium signal transducers. Nat Rev Mol Cell Biol. 13, 549-565 (2012).
- Williams, G. S., Boyman, L., Chikando, A. C., Khairallah, R. J., Lederer, W. J. Mitochondrial calcium uptake. Proc Natl Acad Sci U S A. 110, 10479-10486 (2013).
- Celsi, F., et al. Mitochondria, calcium and cell death: a deadly triad in neurodegeneration. Biochim Biophys Acta. 1787, 335-344 (2009).
- Monteith, G. R., Davis, F. M., Roberts-Thomson, S. J. Calcium channels and pumps in cancer: changes and consequences. J Biol Chem. 287, 31666-31673 (2012).
- Roderick, H. L., et al. Calcium in the heart: when it’s good, it’s very very good, but when it’s bad, it’s horrid. Biochem Soc Trans. 35, 957-961 (2007).
- Grynkiewicz, G., Poenie, M., Tsien, R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 260, 3440-3450 (1985).
- Jin, X., et al. Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli. Cell Mol Life Sci. , (2013).
- Valet, G., Raffael, A., Russmann, L. Determination of intracellular calcium in vital cells by flow-cytometry. Naturwissenschaften. 72, 600-602 (1985).
- June, C. H., Rabinovitch, P. S. Flow cytometric measurement of cellular ionized calcium concentration. Pathology and immunopathology research. 7, 409-432 (1988).
- Ransom, J. T., DiGiusto, D. L., Cambier, J. Flow cytometric analysis of intracellular calcium mobilization. Methods Enzymol. 141, 53-63 (1987).
- Gergely, L., Cook, L., Agnello, V. A simplified method for Ca2+ flux measurement on isolated human B cells that uses flow cytometry. Clin Diagn Lab Immunol. 4, 70-74 (1997).
- Seidel, J., et al. The neurotransmitter GABA is a potent inhibitor of the stromal cell-derived factor-1alpha induced migration of adult CD133+ hematopoietic stem and progenitor cells. Stem cells and development. 16, 827-836 (2007).
- Lee, W. K., Chakraborty, P. K., Roussa, E., Wolff, N. A., Thévenod, F. ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP. Biochim Biophys Acta. 1823, 1864-1876 (2012).
- Woost, P. G., et al. Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats. Kidney Int. 50, 125-134 (1996).
- Beyer, K. H., et al. Benemid,’ p-(di-n-propylsulfamyl)-benzoic acid; its renal affinity and its elimination. Am J Physiol. 166, 625-640 (1951).
- Buckley, B. J., Whorton, A. R. Tunicamycin increases intracellular calcium levels in bovine aortic endothelial cells. Am J Physiol. 273, 1298-1305 (1997).
- Koepsell, H. The SLC22 family with transporters of organic cations, anions and zwitterions. Molecular aspects of medicine. 34, 413-435 (2013).
- Dean, M., Hamon, Y., Chimini, G. The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res. 42, 1007-1017 (2001).
- Brezden, C. B., Hedley, D. W., Rauth, A. M. Constitutive expression of P-glycoprotein as a determinant of loading with fluorescent calcium probes. Cytometry. 17, 343-348 (1994).
- Thévenod, F., Friedmann, J. M., Katsen, A. D., Hauser, I. A. Up-regulation of multidrug resistance P-glycoprotein via nuclear factor-kappaB activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis. J Biol Chem. 275, 1887-1896 (2000).
- Chakraborty, P. K., Lee, W. K., Molitor, M., Wolff, N. A., Thévenod, F. Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells. Mol Cancer. 9, 102 (2010).
- Jennings, L. K., Dockter, M. E., Wall, C. D., Fox, C. F., Kennedy, D. M. Calcium mobilization in human platelets using indo-1 and flow cytometry. Blood. 74, 2674-2680 (1989).
- Lopez, M., Olive, D., Mannoni, P. Analysis of cytosolic ionized calcium variation in polymorphonuclear leukocytes using flow cytometry and Indo-1 AM. Cytometry. 10, 165-173 (1989).
