Lyso-Tracker Red

Lyso-Tracker Red is an eosinophilic red fluorescent probe that can penetrate the cell membrane at neutral pH and can be used for lysosome-specific fluorescent staining of living cells and tracing of acidic organelles. The maximum excitation and emission wavelengths of Lyso-Tracker Red are 577/590 nm^[1-2].

Lyso-Tracker Red is a weakly alkaline fluorescent probe fluorescently labeled with DND-99, in which only the weak base can partially provide protons to maintain the pH at neutral. In the neutral charge state, the Lyso-Tracker Red probe can freely diffuse through the intact plasma membrane of living cells. Due to the inherent acidity in lysosomes, the weakly alkaline groups will be protonated when diffusing into lysosomes. In this charged state, the probe is not easy to diffuse through the organelle membrane, and forms local accumulation in the acidic lysosomes, thereby achieving specific fluorescent labeling of the lysosomes. Lyso-Tracker Red is suitable for fluorescent staining of lysosomes in living cells, but not for fluorescent staining of lysosomes in fixed cells. If cells stained with Lyso-Tracker Red need to be fixed, 3% glutaraldehyde can be tried^[1-2].

Lyso-Tracker Red must be used at very low concentrations to achieve excellent selectivity. Studies of the endocytosis kinetics of the Lyso-Tracker Red probe have shown that the uptake of the dye into living cells takes only seconds. However, these lysosomal probes cause lysosomes to become alkaline, and long-term incubation can induce an increase in lysosomal pH. Therefore, it is recommended that cells should not be incubated with the probe for too long before imaging. When evaluating the number and status of lysosomes in HUVEC cells, acidic compartments in HUVEC can be visualized by staining with Lyso-Tracker Red^[3]. Lysosomal distribution can be monitored by incubating HepG2 cells with 100 nM Lyso-Tracker Red for 30 min^[4]. Furthermore, 100 nM Lyso-Tracker Red can penetrate the MDCK cell membrane at neutral pH and distribute within acidic organelles, such as late endosomes and lysosomes in living cells^[5].

References：
[1] Price O T, Lau C, Zucker R M. Quantitative fluorescence of 5‐FU–treated fetal rat limbs using confocal laser scanning microscopy and Lysotracker Red[J]. Cytometry Part A: The Journal of the International Society for Analytical Cytology, 2003, 53(1): 9-21.
[2] Li G, Ma S, Tang J, et al. Lysosome-targeted two-photon fluorescent probes for rapid detection of H 2 S in live cells[J]. New Journal of Chemistry, 2019, 43(3): 1267-1274.
[3] Wang Y, Bao D, Dong Y, et al. α‐Lipoic Acid‐Plus Ameliorates Endothelial Injury by Inhibiting the Apoptosis Pathway Mediated by Intralysosomal Cathepsins in an In Vivo and In Vitro Endothelial Injury Model[J]. Oxidative Medicine and Cellular Longevity, 2022, 2022(1): 8979904.
[4] Pereira L C, Duarte F V, Varela A T I F, et al. An autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity[J]. Toxicology, 2017, 376: 59-65.
[5] Nagahama M, Itohayashi Y, Hara H, et al. Cellular vacuolation induced by Clostridium perfringens epsilon‐toxin[J]. The FEBS journal, 2011, 278(18): 3395-3407.

Lyso-Tracker Red是一种嗜酸性红色荧光探针，能够在中性pH下穿透细胞膜，可用于活细胞溶酶体特异性荧光染色和酸性细胞器的示踪。Lyso-Tracker Red最大激发波长和最大发射波长为577/ 590 nm^[1-2]。

Lyso-Tracker Red为DND-99进行了荧光标记的弱碱性荧光探针，其中仅弱碱可部分提供质子，以维持pH在中性。在中性电荷状态下，Lyso-Tracker Red探针可自由扩散穿过活细胞的完整质膜。由于溶酶体内固有的酸性，在扩散进入溶酶体时，弱碱性基团会被质子化。在这种带电状态下，探针不易扩散穿过细胞器膜，在偏酸性的溶酶体中形成了局部积累，从而实现对于溶酶体的特异性荧光标记。Lyso-Tracker Red适用于活细胞溶酶体的荧光染色，但不适合用于固定细胞溶酶体的荧光染色，如果经Lyso-Tracker Red染色后的细胞需要进行固定操作，可以尝试3%的戊二醛^[1-2]。

Lyso-Tracker Red必须在极低浓度下才能获得优异的选择性。Lyso-Tracker Red探针的内吞作用动力学研究显示染料进入活细胞的摄入时间仅几秒即可。然而，这些溶酶体探针会导致溶酶体被碱化，长期孵育会诱使溶酶体pH值的增加。因此，建议成像前用探针孵育细胞的时间不能太久。在评估HUVEC细胞中溶酶体的数量和状态时，可通过 Lyso-Tracker Red染色观察HUVEC中的酸性区室^[3]。100 nM Lyso-Tracker Red 孵育HepG2细胞30 min可监测溶酶体分布^[4]。并且，100 nM Lyso-Tracker Red可在中性pH穿透MDCK细胞膜并分布在酸性细胞器内，例如活细胞中的晚期核内体和溶酶体^[5]。