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Measurement of Free Cytosolic Calcium Concentration ([Ca2+]i) in Single CHO-K1 Cells
测定CHO-K1细胞质中的游离钙离子的浓度   

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Abstract

This is a protocol to analyze the functional response of single CHO-K1 cells to a given treatment in terms of changes in free cytosolic calcium concentration ([Ca2+]i). This is possible by using the Ca2+ indicator dye Fura-2 AM, a polyamino carboxylic acid that binds to free intracellular calcium and is excited at 340 nm and 380 nm. The ratio of the emissions at 505 nm after excitation with those wavelengths is directly correlated to the amount of intracellular calcium. This protocol can be applied to other cell types (cell lines or primary cell cultures) by changing the culture conditions accordingly to the cell type.

Keywords: Intracellular pathway(细胞内通路), Microscopy(显微镜), Single cell(单细胞), Cytosolic calcium(Cytosolic钙), Treatment response(治疗反应)

Materials and Reagents

  1. Ca2+ indicator dye Fura-2-acetoxymethyl ester (Fura-2 AM) (Molecular Probes)
  2. Phenol red-free DMEM
  3. NaHCO3

Equipment

  1. 25 mm round glass coverslips
  2. Nikon Eclipse TE200-E microscope with attached back thinned-CCD cooled digital camera (ORCAII BT; Hamamatsu Photonics)

Software

  1. MetaFluor Software (Imaging Corp)

Procedure

  1. Plate 50000 CHO-K1 cells/ml onto 25 mm round glass coverslips for 24 h.
  2. Load the cells with 2.5 μM of the Ca2+ indicator dye Fura-2 AM (Molecular Probes) in phenol red-free DMEM containing 20 mM NaHCO3 (pH 7.4).
  3. Incubate 30 min at 37 °C.
  4. Wash with phenol red-free DMEM and incubate in phenol red-free DMEM containing 20 mM NaHCO3 (pH 7.4) for 15 min (to allow the hydrolysis of the ester group).
  5. Mount the round glass coverslip with the cells on the stage of a Nikon Eclipse TE200-E microscope.
  6. Add 300 μl of phenol red-free DMEM containing 20 mM NaHCO3 (pH 7.4).
  7. Localize the cells to be recorder (as many as possible) and mark them as well as 2-3 background regions using the MetaFluor Software.
  8. Acquire images of the loaded cells under a x40 oil immersion objective during exposure to alternating 340- and 380-nm light beams, and measuring the intensity of light emission at 505 nm every 5 sec.
  9. After 30-40 sec where the baseline is stablished, add the treatment (300 μl) whose effect want to be proven, and continue recording.
  10. Changes in [Ca2+]i after treatment administration are recorded as background substrates ratios of the corresponding excitation wavelength (F340/F380) using MetaFluor Software.

Acknowledgments

This protocol is adapted from Cordoba-Chacon et al. (2010); Cordoba-Chacon et al. (2011) and Duran-Prado et al. (2012).

References

  1. Cordoba-Chacon, J., Gahete, M. D., Duran-Prado, M., Pozo-Salas, A. I., Malagon, M. M., Gracia-Navarro, F., Kineman, R. D., Luque, R. M. and Castano, J. P. (2010). Identification and characterization of new functional truncated variants of somatostatin receptor subtype 5 in rodents. Cell Mol Life Sci 67(7): 1147-1163.
  2. Cordoba-Chacon, J., Gahete, M. D., Pozo-Salas, A. I., Martinez-Fuentes, A. J., de Lecea, L., Gracia-Navarro, F., Kineman, R. D., Castano, J. P. and Luque, R. M. (2011). Cortistatin is not a somatostatin analogue but stimulates prolactin release and inhibits GH and ACTH in a gender-dependent fashion: potential role of ghrelin. Endocrinology 152(12): 4800-4812.
  3. Duran-Prado, M., Bucharles, C., Gonzalez, B. J., Vazquez-Martinez, R., Martinez-Fuentes, A. J., Garcia-Navarro, S., Rhodes, S. J., Vaudry, H., Malagon, M. M. and Castano, J. P. (2007). Porcine somatostatin receptor 2 displays typical pharmacological sst2 features but unique dynamics of homodimerization and internalization. Endocrinology 148(1): 411-421.
  4. Duran-Prado, M., Gahete, M. D., Hergueta-Redondo, M., Martinez-Fuentes, A. J., Cordoba-Chacon, J., Palacios, J., Gracia-Navarro, F., Moreno-Bueno, G., Malagon, M. M., Luque, R. M. and Castano, J. P. (2012). The new truncated somatostatin receptor variant sst5TMD4 is associated to poor prognosis in breast cancer and increases malignancy in MCF-7 cells. Oncogene 31(16): 2049-2061.
  5. Martinez-Fuentes, A. J., Moreno-Fernandez, J., Vazquez-Martinez, R., Duran-Prado, M., de la Riva, A., Tena-Sempere, M., Dieguez, C., Jimenez-Reina, L., Webb, S. M., Pumar, A., Leal-Cerro, A., Benito-Lopez, P., Malagon, M. M. and Castano, J. P. (2006). Ghrelin is produced by and directly activates corticotrope cells from adrenocorticotropin-secreting adenomas. J Clin Endocrinol Metab 91(6): 2225-2231.

简介

这是用于分析单个CHO-K1细胞对给定治疗的功能性反应的方案,其根据游离胞质钙浓度([Ca 2+] i)的变化。 这是可能的,通过使用Ca2 +指示剂染料Fura-2AM,一种聚氨基羧酸,其结合游离细胞内钙,并在340nm和380nm激发。 在用那些波长激发后在505nm的发射的比率与细胞内钙的量直接相关。 该方案可以通过根据细胞类型改变培养条件而应用于其它细胞类型(细胞系或原代细胞培养物)。

关键字:细胞内通路, 显微镜, 单细胞, Cytosolic钙, 治疗反应

材料和试剂

  1. Ca 2+指示剂染料Fura-2-乙酰氧基甲基酯(Fura-2AM)(Molecular Probes)
  2. 酚红无色DMEM
  3. NaHCO 3

设备

  1. 25毫米圆形玻璃盖片
  2. 附带背面薄型CCD冷却数码相机(ORCAII BT; Hamamatsu Photonics)的Nikon Eclipse TE200-E显微镜

软件

  1. MetaFluor软件(Imaging Corp)

程序

  1. 将50000个CHO-K1细胞/ml板置于25mm圆形玻璃盖玻片上24小时
  2. 用2.5μM的Ca 2+指示剂染料Fura-2AM(Molecular Probes)在含有20mM NaHCO 3(pH 7.4)的无酚红DMEM中加载细胞, 。
  3. 在37℃孵育30分钟。
  4. 用无酚红DMEM洗涤并在含有20mM NaHCO 3(pH 7.4)的无酚红DMEM中孵育15分钟(以允许酯基水解)。
  5. 将圆玻璃盖玻片与细胞放在Nikon Eclipse TE200-E显微镜的镜台上
  6. 加入300μl含有20mM NaHCO 3(pH 7.4)的不含酚红的DMEM。
  7. 本地化要记录的单元格(尽可能多),并使用MetaFluor软件将其标记为2-3个背景区域。
  8. 在曝光期间在交替的340和380nm光束下获取装载的细胞在x40油浸物镜下的图像,并且每5秒测量505nm处的光发射强度。
  9. 在基线稳定的30-40秒后,添加其效果需要证实的治疗(300μl),然后继续记录。
  10. 使用MetaFluor软件记录治疗施用后[Ca 2+ 2 +]/- 的变化作为相应激发波长(F340/F380)的背景底物比例。

致谢

该协议改编自Cordoba-Chacon等人(2010); Cordoba-Chacon等人(2011)和Duran-Prado等人(2012)。

参考文献

  1. Cordoba-Chacon,J.,Gahete,M.D.,Duran-Prado,M.,Pozo-Salas, Malagon,M.M.,Gracia-Navarro,F.,Kineman,R.D.,Luque,R.M Castano,J.P。(2010)。 在啮齿动物中生长抑素受体亚型5的新功能性截短变体的鉴定和表征。 em> Cell Mol Life Sci 67(7):1147-1163。
  2. 科尔多瓦 - 查康,J.,Gahete,MD,Pozo-Salas,AI,Martinez-Fuentes,AJ,de Lecea,L.,Gracia-Navarro,F.,Kineman,RD,Castano,JP和Luque,RM(2011) 。 皮质抑素不是生长抑素类似物,但刺激催乳素释放,并以性别依赖性方式抑制GH和ACTH :ghrelin的潜在作用。内分泌学。 152(12):4800-4812。
  3. Duran-Prado,M.,Bucharles,C.,Gonzalez,BJ,Vazquez-Martinez,R.,Martinez-Fuentes,AJ,Garcia-Navarro,S.,Rhodes,SJ,Vaudry,H.,Malagon, ,JP(2007)。 猪生长抑素受体2显示典型的药理学sst2特征,但具有同源二聚化和内化的独特动力。 Endocrinology 148(1):411-421。
  4. Duran-Prado,M.,Gahete,M.D.,Hergueta-Redondo,M.,Martinez-Fuentes, A.C.,Cordoba-Chacon,J.,Palacios,J.,Gracia-Navarro,F., Moreno-Bueno,G.,Malagon,M.M.,Luque,R.M.and Castano,J.P。 (2012)。 新的截短的生长抑素受体变体sst5TMD4是相关的 乳腺癌的预后不良和增加MCF-7细胞的恶性肿瘤。癌基因 31(16):2049-2061。
  5. Martinez-Fuentes,AJ,Moreno-Fernandez,J.,Vazquez-Martinez,R.,Duran-Prado,M.,de la Riva,A.,Tena-Sempere,M.,Dieguez,C.,Jimenez-Reina, L.,Webb,SM,Pumar,A.,Leal-Cerro,A.,Benito-Lopez,P.,Malagon,MM和Castano,JP(2006)。 Ghrelin是由促肾上腺皮质激素分泌性腺瘤产生并直接激活促肾上腺皮质激素细胞。 J Clin Endocrinol Metab 91(6):2225-2231
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引用:Gahete, M. D., Luque, R. M. and Castaño, J. P. (2012). Measurement of Free Cytosolic Calcium Concentration ([Ca2+]i) in Single CHO-K1 Cells. Bio-protocol 2(22): e294. DOI: 10.21769/BioProtoc.294.
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