Image of Molecular Probes® DAPI packaging

Nuclear stain for HCA/HCS cell demarcation

A popular nuclear and chromosome counterstain, DAPI (4′,6-diamidino-2-phenylindole) emits blue fluorescence upon binding to AT regions of DNA. This protocol produces sufficient stain for one 96-well plate at a staining volume of 100 μL/well.

This protocol can be used for:

  • Nuclear demarcation in high-content analysis/screening (HCA/HCS)

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:


Preparing solutions

1. Add 2 mL deionized water (diH2O) or dimethylformamide (DMF) to the entire contents of the DAPI vial to make a 14.3 mM (5 mg/mL) DAPI stock solution. Note: DAPI has poor solubility in water, so sonicate as necessary to dissolve. The 5 mg/mL DAPI stock solution may be stored at 2–6°C for up to 6 months or at ≤–20°C for longer periods.
2. Add 2.1 µL of the 14.3 mM DAPI stock solution to 100 µL PBS to make a 300 µM DAPI intermediate dilution.
3. Add 10 µL of the 300 µM DAPI intermediate dilution to 10 mL PBS to make a 300 nM DAPI stain solution.

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 100 μL 300 nM DAPI stain solution to each well.
3. Incubate for 1–5 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
Excitation/Emission (nm)358/461
Standard filter setDAPI
Storage conditions2–6°C or ≤–20°C



Protocol tips

  • DAPI is a known mutagen and should be handled with care.

Cells stained with DAPI
Cells stained with DAPI and imaged with the Thermo Scientific CellInsight High-Content System.