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.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.