Key Laboratory of Laser Life Science, Ministry of Education, College of Biophotonics, South China Normal University, Guangzhou, China.
Guangdong Key Laboratory of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
Cytometry A. 2021 Jun;99(6):632-640. doi: 10.1002/cyto.a.24316. Epub 2021 Feb 12.
Three-cube Förster resonance energy transfer (FRET) method is the most extensively applied approach for live-cell FRET quantification. Reliable measurements of calibration factors are crucial for quantitative FRET measurement. We here proposed a modified TA-G method (termed as mTA-G) to simultaneously obtain the FRET-sensitized quenching transition factor (G) and extinction coefficients ratio (γ) between donor and acceptor. mTA-G method includes four steps: (1) predetermining the ratio ranges of the sensitized emission of acceptor (F ) to the donor excitation and donor channel image (I [(DA])) for all FRET plasmids; (2) culturing the cells which express every FRET plasmid in one dish respectively; (3) distinguishing and marking the cells expressing different FRET plasmids by detecting their F /I (DA) values; (4) linearly fitting F /I (DA) (acceptor excitation and acceptor channel image) to I (DA)/I (DA) for different kinds of cells. We implemented mTA-G method by imaging tandem constructs cells with different FRET efficiency cultured in one dish on different days, and obtained consistent G and γ values. mTA-G method not only circumvents switchover of different culture dishes but also keep the constant imaging conditions, exhibiting excellent robustness, and thus will expands the biological applications of quantitative FRET analysis in living cells.
三晶球Förster 共振能量转移（FRET）方法是最广泛应用于活细胞 FRET 定量的方法。可靠的校准因子测量对于定量 FRET 测量至关重要。我们在这里提出了一种改进的 TA-G 方法（称为 mTA-G），用于同时获得 FRET 敏化猝灭跃迁因子（G）和供体与受体之间的消光系数比（γ）。mTA-G 方法包括四个步骤：（1）预先确定所有 FRET 质粒的敏化受体发射（F）与供体激发和供体通道图像（I [(DA)]）之间的比值范围；（2）分别在一个培养皿中培养表达每种 FRET 质粒的细胞；（3）通过检测它们的 F / I （DA）值来区分和标记表达不同 FRET 质粒的细胞；（4）用不同种类的细胞线性拟合 F / I （DA）（受体激发和受体通道图像）与 I （DA）/ I （DA）。我们通过在不同天对在一个培养皿中培养的具有不同 FRET 效率的串联构建细胞进行成像，实施了 mTA-G 方法，并获得了一致的 G 和γ值。mTA-G 方法不仅避免了不同培养皿的切换，而且保持了恒定的成像条件，表现出出色的稳健性，从而将定量 FRET 分析在活细胞中的生物学应用扩展。
