PKH67 is a fluorescent cell-binding dye with green fluorescence, capable of staining cell membranes, with excitation/emission (Ex/Em) at 490/502 nm. It is often used in combination with the non-specific red fluorescent dye PKH26 (Ex/Em=551/567 nm) to label cells, detect cell proliferation in vitro, and trace cells both in vitro and in vivo [1] [2].

PKH 67: To prepare the staining solution, first take the PKH 67 reagent from the refrigerator and allow it to reach room temperature, or briefly heat it in a 37°C water bath. Centrifuge the tube containing PKH 67 before opening to ensure the reagent settles at the bottom. Depending on the number of cell samples to be tested, dilute the probe by 10 times with a diluent, and further dilute the PKH 67 stock solution 25 times with a suitable solution (e.g., protein-free media, HBSS, or PBS) to create the staining working solution. Adjust the optimal working solution concentration based on the type of cells and experimental system; generally, a 250-fold dilution of the stock solution is sufficient, although some cells may require a higher concentration. For cell staining, resuspend prepared cells in 100 μL of the staining working solution to achieve a cell concentration of approximately 10^7/mL or perform in situ staining by covering cells with enough staining solution. Culture the cells at 2-8°C for 15-30 minutes, adjusting incubation time according to cell type. Note that incubating cells at 37°C for 5 minutes followed by 4°C for 15 minutes is recommended to reduce dye endocytosis and enhance membrane labeling, minimizing potential dye localization in cytosolic vesicles. After incubation, centrifuge the cells to remove the supernatant, wash the cells 1-2 times with PBS or protein-free media, and resuspend them in PBS or protein-free media. Use 500 μL of cell suspension for flow cytometry analysis (Ex/Em = 490/502 nm). Subsequently, cells can be cultured according to normal methods, directly observed using a fluorescence microscope, or the proliferation of cells can be assessed after appropriate culture time using flow cytometry or other specific experimental objectives for cell fluorescence tracing. It is noteworthy that staining concentration varies depending on cell type and cell number per well. PKH 67 stock solution is prone to hydrolysis; hence, it should be aliquoted and stored frozen at ≤-20°C. The PKH 67 working solution should be prepared fresh for immediate use, as PKH 67 absorbs moisture and decomposes, affecting staining results. Avoid water contact during usage, although brief contact with water during cell marking is permissible. PKH 67, an ethanol solution, solidifies at lower temperatures such as 4°C or in ice baths, sticking to the tube bottom, wall, or lid. Allow it to reach room temperature to liquefy before centrifuging to the tube bottom. If needed, briefly use a 37°C water bath to fully liquefy before use. The tracing capability of labeled cells varies significantly with cell type and period, so refer to actual conditions or relevant literature for evaluation.