DeepRed Nucleus Dye

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
Mammalian cell in full culture medium staining methods^[2]:
(1) Cell planking: Digestive separation of cells and resuspend in complete medium to a concentration of 2-4 × 10⁵ cells/mL.
Note: Attached cell cultures (e.g., coverslip cultures or chambered wells) can be stained in a 1-2-mL staining volume overlayering a 4-cm² surface area.
(2) Prepare staining solution: Add 4 μL of 5 mM DeepRed Nucleus Dye acidified stock per ml culture medium (20 μM final).
Note: Nuclear discrimination is achievable at 2.5 to 5 μM, and it is unlikely that concentrations >30 μM would be required.
(3) Fluorescence staining: Incubate 5 to 15 min at 37°C.
Note: Overstaining cannot occur.
(4) Wash (optional): Centrifuge cells 3 to 5 min at 800 × g, 37°C. Discard supernatant and resuspend in complete medium with 10 mM HEPES (HY-D0857) at 4 × 10⁵ cells/mL .
(5) For flow cytometry: Use conventional pulse analysis for doublet discrimination and analyze parameters using appropriate software.
(6) For laser scanning microscopy: Collect fluorescence images using a 695 nm long-pass filter.
Fixed cells staining methods^[2]:
(1) Fixed cells: Use 4% paraformaldehyde in PBS for 30 min with resuspension in an aqueous buffer (e.g., PBS).
(2) Fluorescence staining: similar concentrations of dye and similar incubation conditions can be used as for live cells.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.