验证基因编码的Ca2+传感器的功能和定位，并将荧光共振能量转移（FRET）比率转换为Ca2+浓度。
Verifying the function and localization of genetically encoded Ca2+ sensors and converting FRET ratios to Ca2+ concentrations.
作者信息
Park J Genevieve, Palmer Amy E
机构信息
Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, Colorado 80309.
出版信息
Cold Spring Harb Protoc. 2015 Jan 5;2015(1):pdb.prot076547. doi: 10.1101/pdb.prot076547.
Abstract
Genetically encoded, ratiometric, fluorescent Ca(2+) biosensors can be used in living cells to quantitatively measure free Ca(2+) concentrations in the cytosol or in organelles. This protocol describes how to perform a calibration of a Ca(2+) sensor expressed in cultured mammalian cells as images are acquired using a widefield fluorescence microscope. This protocol also explains how to calculate Förster resonance energy transfer (FRET) ratios from acquired images and how to convert FRET ratios to Ca(2+) concentrations.
摘要
基因编码的、比率型荧光钙生物传感器可用于活细胞，以定量测量细胞质或细胞器中的游离钙离子浓度。本实验方案描述了在使用宽场荧光显微镜采集图像时，如何对培养的哺乳动物细胞中表达的钙传感器进行校准。本实验方案还解释了如何从采集的图像中计算福斯特共振能量转移（FRET）比率，以及如何将FRET比率转换为钙离子浓度。
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本文引用的文献
1
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PLoS One. 2011;6(12):e29057. doi: 10.1371/journal.pone.0029057. Epub 2011 Dec 27.
2
Mechanisms of control of the free Ca2+ concentration in the endoplasmic reticulum of mouse pancreatic β-cells: interplay with cell metabolism and [Ca2+]c and role of SERCA2b and SERCA3.
Diabetes. 2011 Oct;60(10):2533-45. doi: 10.2337/db10-1543. Epub 2011 Sep 1.
3
Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn2+ with genetically encoded sensors.
Proc Natl Acad Sci U S A. 2011 May 3;108(18):7351-6. doi: 10.1073/pnas.1015686108. Epub 2011 Apr 18.
4
Reticulon short hairpin transmembrane domains are used to shape ER tubules.
Traffic. 2011 Jan;12(1):28-41. doi: 10.1111/j.1600-0854.2010.01134.x. Epub 2010 Nov 12.
5
Spontaneous network activity visualized by ultrasensitive Ca(2+) indicators, yellow Cameleon-Nano.
Nat Methods. 2010 Sep;7(9):729-32. doi: 10.1038/nmeth.1488. Epub 2010 Aug 8.
6
Using a genetically targeted sensor to investigate the role of presenilin-1 in ER Ca2+ levels and dynamics.
Mol Biosyst. 2010 Sep;6(9):1640-9. doi: 10.1039/c001975e. Epub 2010 Apr 8.
7
A genetically encoded calcium indicator for chronic in vivo two-photon imaging.
Nat Methods. 2008 Sep;5(9):805-11. doi: 10.1038/nmeth.1243.
8
Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs.
Chem Biol. 2006 May;13(5):521-30. doi: 10.1016/j.chembiol.2006.03.007.
9
Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor.
Proc Natl Acad Sci U S A. 2004 Dec 14;101(50):17404-9. doi: 10.1073/pnas.0408030101. Epub 2004 Dec 7.
10
Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.
Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10554-9. doi: 10.1073/pnas.0400417101. Epub 2004 Jul 9.
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