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培养细胞中游离钙和总细胞内钙的定量成像

Quantitative imaging of free and total intracellular calcium in cultured cells.

作者信息

Chandra S, Gross D, Ling Y C, Morrison G H

机构信息

Department of Chemistry, Cornell University, Ithaca, NY 14853.

出版信息

Proc Natl Acad Sci U S A. 1989 Mar;86(6):1870-4. doi: 10.1073/pnas.86.6.1870.

DOI:10.1073/pnas.86.6.1870
PMID:2928310
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC286806/
Abstract

Techniques of fluorescence and ion microscopies were combined to study the free [Ca2+] and total Ca in NIH 3T3 fibroblast and L6 rat myoblast cells. Free Ca2+ measurements with the Ca2+ indicator fura-2 and digital imaging reveal an inhomogeneous distribution of free cytoplasmic Ca2+ in both cell lines. Fura-2 also reveals a difference in free Ca2+ activity between the nucleus and cytoplasm of cells. Ion microscopic observations on sister cells show that total Ca in the cytoplasm is also inhomogeneously distributed and that mean cytoplasmic levels of total Ca are higher than levels in the nuclei. In the nuclei of NIH 3T3 cells, the mean free [Ca2+] and total [Ca] were 110 +/- 30 nM and 225 +/- 43 microM, respectively, while regions in the cell cytoplasm contained up to 490 +/- 270 nM free [Ca2+] and 559 +/- 184 microM mean total [Ca]. Intracellular total Ca was greater than 3 orders of magnitude higher than intracellular free Ca2+ in either nuclear or cytoplasmic compartments. Perinuclear cytoplasmic regions in 3T3 cells contained higher free and total Ca than the cell nucleus. Loading of cells with fura-2 did not modify the subcellular distribution of total K, Na, Ca, or Mg. This combination of two powerful ion imaging techniques provides a comparison between free and total calcium in cells and introduces a different approach for examining the role of this important element in cell physiology.

摘要

荧光显微镜技术和离子显微镜技术相结合，用于研究NIH 3T3成纤维细胞和L6大鼠成肌细胞中的游离[Ca2+]和总钙含量。使用Ca2+指示剂fura-2和数字成像技术对游离Ca2+进行测量，结果显示两种细胞系中细胞质内游离Ca2+的分布均不均匀。Fura-2还揭示了细胞核与细胞质之间游离Ca2+活性的差异。对姐妹细胞进行离子显微镜观察发现，细胞质中的总钙分布也不均匀，且细胞质中总钙的平均水平高于细胞核中的水平。在NIH 3T3细胞的细胞核中，游离[Ca2+]和总[Ca]的平均值分别为110±30 nM和225±43 μM，而细胞质区域中游离[Ca2+]高达490±270 nM，总[Ca]的平均水平为559±184 μM。在细胞核或细胞质区室中，细胞内总钙比细胞内游离Ca2+高3个数量级以上。3T3细胞的核周细胞质区域中游离钙和总钙含量均高于细胞核。用fura-2加载细胞不会改变细胞内总钾、钠、钙或镁的亚细胞分布。这两种强大的离子成像技术相结合，可对细胞内的游离钙和总钙进行比较，并为研究这一重要元素在细胞生理学中的作用引入了一种不同的方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c2/286806/2f0e01ee523d/pnas00246-0141-g.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c2/286806/4c6e39d27d3c/pnas00246-0140-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c2/286806/b2da0de09cc7/pnas00246-0140-d.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c2/286806/8cadc30b5ed9/pnas00246-0141-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c2/286806/b8bedc271552/pnas00246-0141-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c2/286806/ecdd184c8018/pnas00246-0141-c.jpg
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