Measurement of the Transfection Efficiency of Cultivated Cells

化学转染

With chemical transfection, the negative charge of the nucleic acid is made positive or neutralized. Methods include lipofection and calcium phosphate co-precipitation, with lipofection being the most common.
LiPofection方法涉及通过带正电的合成脂质试剂头组和带负电荷的核酸之间的静电相互作用来构建阳离子脂质试剂复合物。阳离子脂质试剂复合物与负电荷的细胞膜表面相互作用，有效地将核酸引入细胞中。这使得细胞可以通过内吞作用在细胞质中吸收核酸。LiPofection是一种简单简单的转染方法，具有低毒性和出色的可操作性。
This method enables efficient uptake of both nucleic acids, DNA, and RNA. The lipofection method can also be used for both transient and stable transfections.
For chemical transfection, however, the conditions must be appropriate for the cell type and culture, resulting in a lower transfection efficiency than physical and biological methods.

生物转染

生物转染方法涉及病毒。基于病毒的转染具有高效，因为它利用了病毒的感染性。该方法用于对核酸摄取的细胞中蛋白质的过表达。该方法是体内转染的理想选择，使其成为临床研究最常用的方法。腺病毒，逆转录病毒和慢病毒用于体内基因摄取。
通过生物转染，在基因复制过程中会产生重组病毒，并在仅提取病毒载体之前用具有辅助功能的基因转染的细胞被接受并传播。然后生成包含要掺入基因的病毒载体，以将所需的遗传物质传递到细胞。
尽管基于病毒的转染具有高度适应活性生物的适应性，例如用于动物实验，但准备病毒可能很困难，并且需要遵守生物安全标准。免疫系统的插入突变和失活也有问题。

Physical transfection

Physical transfection involves stimulating the surface of the cell to introduce the nucleic acid through the cell membrane. Methods include electroporation, microinjection, and laser processing, with electroporation being the most common. During electroporation, the nucleic acids and the cells are suspended in a conductive solution and then clamped between an anode and a cathode. Electric pulses are then applied continuously to create small holes in the cell membrane. These holes are then used to transfer extracellular nucleic acids with an electrical charge—such as DNA and RNA—through the cell membrane.
物理转染具有较高的转染效率，并且比化学和生物转染方法更容易。但是，电脉冲可能会导致细胞死亡，电脉冲制成的孔可能不会接近，导致细胞毒性，并且需要昂贵的专用设备。

转染效率测量

在基因引入期间，将用生长培养基制备的细胞悬浮液播种，以适当的细胞密度进入井板并孵化。然后，将制备的复合物添加到细胞中，摇动井板，然后在孵化器中再次孵育24小时。当孵育结束时，观察细胞的表达以测量转染效率。通常使用荧光染料和荧光显微镜测量转染效率。

转染效率测量挑战

Conventional fluorescence microscopes pose various problems and are often difficult to use.
首先，观察荧光需要将样品带到暗室并在那里执行实验，从而导致可操作性极差。精确提取细胞轮廓也很困难，阻碍了细胞数量的准确计数。
此外，为了计数和分析单元格的数量，需要与荧光显微镜系统分开的软件，从而导致许多客户注意执行平滑分析的困难。

The BZ-X800 All-in-One Fluorescence Microscope includes a built-in specimen enclosure that allows users to perform fluorescence imaging in a brightly lit room. The BZ-X800 accurately extracts and quantifies phase-contrast image contours, even for cultured cells with subtle luminance differences, and allows users to specify a cell mask area for measuring the number of measurement targets and the surface area ratio. The BZ-X800 can also measure cell expression levels for quantitative measurement of transfection efficiency without needing separate software.