PKH 26 is a red-fluorescent membrane dye, Ex/Em=551/567 nm.PKH 26 is commonly used in flow cytometry to measure T cell-mediated cytotoxic activity.

Instructions
I. Preparation of stock solution
1. Preparation of mother solution: Dissolve PKH26 in DMSO to prepare a 1 mM stock solution.
2. Preparation of working solution: Dilute the PKH26 stock solution to the working concentration using the same diluent. The common final concentration is 2 × 10⁻⁶ M.
II. Operation steps
1. Cell preparation Collect cells and wash with serum-free medium or PBS to remove residual proteins that may interfere with staining; resuspend cells in an appropriate diluent serum-free medium or PBS.
2. Staining steps
1) Mix equal volumes of cell suspension and dye solution to achieve the desired final dye concentration.
2) Incubate at room temperature for 20–50 minutes, shaking gently to ensure uniform staining.
3. Stop staining
Discard the staining solution, wash 2~3 times with PBS, and resuspend cells in serum-free cell culture medium or PBS. Observe by fluorescence microscopy or flow cytometry.
4. Analysis
After staining, assess cell viability and fluorescence intensity using an appropriate method such as flow cytometry.