EthD-1 (Ethidium homodimer) is a high-affinity fluorescent nucleic acid dye that emits red fluorescence upon binding to DNA or RNA, with excitation/emission wavelengths of 528/617 nm. EthD-1 is impermeable to cells and cannot be used in live cells.

Instructions for the Use of EthD-1 (This protocol is for reference only and should be adjusted according to your specific experiment):
1. Stock Solution Preparation: Prepare a 2 mM EthD-1 stock solution using DMSO. Aliquot and store at -20°C or -80°C, protected from light. Avoid repeated freeze-thaw cycles.
2. Working Solution Preparation Dilute the stock solution with pre-warmed serum-free cell culture medium or PBS to a final concentration of 0.1–10 μM.
Note: The optimal working concentration should be determined based on cell type and experimental requirements. The working solution must be prepared fresh before use.
3. Cell Preparation Suspension cells: Collect by centrifugation, wash twice with PBS (5 minutes each), and remove residual medium. Adherent cells: Discard the culture medium, detach cells using trypsin to create a single-cell suspension, centrifuge to remove supernatant, then wash twice with PBS (5 minutes each). Note: If not performing flow cytometry, adherent cells can be stained on coverslips.
4. Staining Incubation Add 1 mL of EthD-1 working solution and incubate at room temperature for 15–60 minutes, protected from light.
Note: Prolonged incubation may lead to non-specific binding; it is recommended to optimize the incubation time via preliminary experiments.
5. Washing and Resuspension Centrifuge at 400 g for 3–4 minutes at 4°C to remove the staining solution. Wash cells twice with PBS (5 minutes each), then resuspend the cell pellet in 1 mL of serum-free culture medium or PBS.
6. Detect using fluorescence microscopy or flow cytometry.