Although Rhod-2 is the most popular red fluorescent Ca2+probe, its mitochondrial localization and high basal Ca2+signaling in cells severely limit its application in certain cellular settings. In addition, the unsatisfactory excitation wavelength of Rhod-2 at 488nm makes its signal weak on some instruments with only 488nm excitation light sources, such as FLIPRTM. Rhod-4 was improved based on these defects and has the following characteristics:
1) The maximum excitation and emission wavelengths of Rhod-4 are 530nm and 555nm. Although the maximum excitation wavelength of Rhod-4 is 530nm, it is at 488nm
The signal under excitation is also quite strong (see Fig1). In addition to being able to excite at long wavelengths of 514nm, 532nm, and 546nm, Rhod-4 can also obtain ideal fluorescence signals under 488nm excitation using a xenon ion exciter. Rho
The fluorescence signal of d-4 increases with increasing Ca2+concentration (555nm emission wavelength), and once combined with Ca2+, the fluorescence increases by more than 200 times. Due to the fact that the Ca2+concentration in many cells increases by 5-10 times after stimulation
Rhod-4 is an excellent high-sensitivity probe for detecting changes in Ca2+concentration within this range.
2) Once stimulated by agonists, the fluorescence of Rhod-4 in cells is significantly stronger than that of Rhod-2 (signal-to-noise ratio). More importantly, Rhod-4 is mainly located in the cytoplasm of cells, unlike Rhod-2 which is mainly located in mitochondria. another
Furthermore, Rhod-4 exhibits lower temperature dependent cell loading characteristics, producing similar results at both room temperature and 37 ℃. This feature makes Rhod-4 more advantageous than Rhod-2 in HTS applications.
Rhod-4, AM is an acetyl methyl derivative of Rhod-2, which has cell membrane permeability and can easily enter cells with simple cultivation. Once inside the cell, Rhod-4 is cleaved by its lactonase to produce non membrane permeable Rhod-4, which remains in the cell to perform its corresponding physiological functions. This product is provided in the form of freeze-dried powder. When used, it only needs to be fully dissolved in anhydrous DMSO, prepared into a storage solution of 2-5mM, and adjusted to the required working concentration based on specific experiments and relevant literature.
Rhod-2虽然是最受欢迎的红色荧光Ca2+荧光探针，但Rhod-2的线粒体定位和细胞中高基底Ca2+信号使其在某些细胞应用中严重受限制。另外，Rhod-2在488nm处很不理想的激发波长使其在某些只有488nm激发光源的仪器比如FLIPRTM上信号很弱。Rhod-4正是根据这些缺陷而改进的，具有以下特征:
1) Rhod-4的最大激发和发射波长是530nm和555nm。虽Rhod-4最大激发波长是530nm，但在488nm处激发下的信号也相当强。除了能在514nm，532nm和546nm的长波长激发之外，Rhod-4用氙离子激发器在488nm激发下也能得到理想的荧光信号。Rhod-4随着增加的Ca2+浓度荧光信号而增强(555nm发射波长)，一旦与Ca2+结合荧光约增强>200倍。由于许多细胞受刺激后的Ca2+浓度提高通常是5~10倍，因此，Rhod-4是一款优秀的检测此范围内Ca2+浓度变化的高灵敏探针。
2) 一旦受激动剂刺激后，Rhod-4在细胞内的荧光明显强于Rhod-2(信噪比)。更重要的是，Rhod-4主要定位在细胞胞浆内，不像Rhod-2主要定位在线粒体。另外，Rhod-4具更低的温度依赖性的细胞加载特性，不管室温还是37℃产生相似的结果。这一特征使Rhod-4比Rhod-2在HTS应用中更有优势。
Rhod-4,AM是Rhod-2的一种乙酰甲酯衍生物，具有细胞膜渗透性，只需简单培养，即可轻易进入细胞。一旦进入细胞内，即被其内酯酶剪切生成不具膜渗透性的Rhod-4，从而滞留在胞内以发挥相应生理功能。本品以冻干粉的形式提供，使用时只需经无水DMSO充分溶解，配置成2~5mM的储存液，并依据具体实验和相关文献资料调整到需要的工作浓度即可。