Can DAPI be used on live cells?
DAPI is generally used to stain fixed cells since the dye is cell impermeant, although the stain will enter live cells when used at higher concentrations. For live-cell staining, Hoechst 33342 dye is a popular cell-permeant nuclear counterstain.
What concentration is DAPI used at?
It is recommended to use DAPI Staining Solution at a final concentration of 0.1 µg/mL. Since application vary, each investigator should titrate the reagent to obtain optimal results. For dead cell exclusion add 10 µL of DAPI Staining Solution to 10⁶ cells in 1 mL buffer and proceed directly to flow cytometric analysis.
What is DAPI diluted in?
Counterstaining protocol Dilute the DAPI stock solution to 3 µM in staining buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% Nonidet P-40). A 1 mL volume will be required for each cell sample.
How does DAPI stain live cells?
How to Stain Live Cells
- Add the dye to complete culture medium. Use Hoechst dyes at 1 ug/mL or DAPI at 10 ug/mL.
- Remove culture medium from the cells and replace with medium containing dye.
- Incubate cells at room temperature or 37°C for 5-15 minutes, then image.
Does DAPI stain live or dead cells?
DAPI and PI only inefficiently pass through an intact cell membrane and, therefore, preferentially stain dead cells. TMRM is a cell-permeable fluorescent dye that is sequestered to active mitochondria, and hence labels live cells.
How do you make DAPI solution?
To make a 5 mg/mL DAPI stock solution (14.3 mM for the dihydrochloride or 10.9 mM for the dilactate), dissolve the contents of one vial (10 mg) in 2 mL of deionized water (dH2O) or dimethylformamide (DMF).
What is the concentration of DAPI for immunofluorescence?
between 1 – 0.1 µg/ml
Immunofluorescence: Counterstain with DAPI as the final step in your staining procedure. Rinse samples twice in PBS for five min each. Dilute DAPI stock solution to a concentration between 1 – 0.1 µg/ml in PBS and incubate for 5 min at room temperature in the dark.
How do you prepare DAPI solution?
Preparing the DAPI stock solution To make a 5 mg/mL DAPI stock solution (14.3 mM for the dihydrochloride or 10.9 mM for the dilactate), dissolve the contents of one vial (10 mg) in 2 mL of deionized water (dH2O) or dimethylformamide (DMF).
What structure or molecule will the DAPI stain in the cell?
DAPI (pronounced ‘DAPPY’, /ˈdæpiː/), or 4′,6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA.
How do you use DAPI in flow cytometry?
Tap to loosen the pellet and add 1 mL of DAPI diluted in staining buffer. Incubate for 15 minutes at room temperature. Analyze by flow cytometry in the presence of the dye. If the cells are to be viewed by fluorescence microscopy, centrifuge the sample, remove the supernatant and resuspend cells in fresh buffer.
How is DAPI used in live cell staining?
DAPI (4′,6-diamidino-2-phenylindole) DAPI is generally used to stain fixed cells since the dye is cell impermeant, although the stain will enter live cells when used at higher concentrations. For live-cell staining, Hoechst 33342 dye is a popular cell-permeant nuclear counterstain.
How much DAPI to incubate FACS cells?
For having used DPAI in FACS and IHC, 1µg/mL works nicely. If you are working with adherent cells, you can use 1µg/mL of DAPI in PBS. Use minimum volume, just to cover the cells and incubate for 15 minutes in dark followed by three time washing with PBS .
How to use DAPI for fluorescence imaging of embryos?
1. Wash the cells 1–3 times in PBS as needed. 2. Add sufficient 300 nM DAPI stain solution to cover the cells. 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. DAPI is a known mutagen and should be handled with care. Drosophila melanogaster embryo staining.
What’s the best concentration of DAPI to use?
DAPI is one of those stains that works really well at a range of concentrations. You can use it anywhere from 0.1µg/ml 10µg/ml Generally, 10mins in the dark at room temperature is adequate