DAPI Protocol for HCA Instruments
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:
- Cells growing in culture
- DAPI, dihydrochloride (Cat. No. D1306)
- Flat-bottom 96-well microplates
- Phosphate-buffered saline (PBS)
- HCA instrument
|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.|
|Standard filter set||DAPI|
|EVOS® Light Cube||DAPI|
|Storage conditions||2–6°C or ≤–20°C
- DAPI is a known mutagen and should be handled with care.
Cells stained with DAPI and imaged with the Thermo Scientific™ CellInsight™ High-Content System.