NucRed Dead 647 ReadyProbes Reagent Protocol for Fixed Cells

image of Molecular Probes® NucRed® Dead 647 ReadyProbes® Reagent

Nuclear stain for fixed cells

NucRed Dead 647 ReadyProbes Reagent emits bright far-red fluorescence when bound to DNA and is suitable for staining nuclei in fixed-cell preparations and tissue sections. NucRed Dead 647 reagent may also be used to measure viability and to measure cytotoxicity in kinetic live-cell assays.

This protocol can be used for:

Nuclear staining to identify nuclear condensation in apoptotic cells using fluorescence microscopy

This protocol should not be used for:

Flow cytometry

You will need the following for this protocol:

Cells growing in culture
NucRed Dead 647 ReadyProbes Reagent (Cat. No. R37113)
Fluorescence microscope with far red or Cy5 filters

Protocol

Labeling fixed cells

First, fix and permeabilize cultured cells with a protocol appropriate for your sample.

1. Wash the cells 1–3 times in PBS as needed.

2. Add two drops of NucRed Dead 647 Reagent per milliliter of medium.

3. Incubate for 5–15 minutes, protected from light.

4. Remove the stain solution.

5. Wash the cells 2–3 times in PBS.

6. Image the cells.

Spectral information and storage

	NucRed
Excitation/Emission (nm)	642/661
Standard filter set	Cy5
EVOS Light Cube	Cy5
Storage conditions	Room temperature

Protocol tips

In some cases, more or fewer drops may be needed to achieve optimal staining intensity.
Image quality may be improved by replacing the culture medium with Live Cell Imaging Solution (Cat. No. A14291DJ).

Tubulin and nuclear staining in HeLa cells. HeLa cells were fixed and permeabilized and stained for tubulin (biotinylated mouse anti-a-tubulin mouse monoclonal antibody followed by an Alexa Fluor 488 goat anti-mouse secondary antibody) and counter-stained with NucRed Dead 647 ReadyProbes reagent.

For Research Use Only. Not for use in diagnostic procedures.