DAPI Nuclear Stain, 10mg

Stain nuclear DNA with this bright blue fluorescent dye to assess nuclear content or counterstain cells for visualization by fluorescence microscopy.

Staining Adherent Cells for Fluorescence Microscopy:

1. If fixing cells, use the fixation protocol appropriate for your sample. DAPI staining is normally performed after fixation and all other staining.
2. Equilibrate the sample briefly with phosphate-buffered saline (PBS).
3. Dilute the DAPI stock solution to 300 nM in PBS.
4. Add approximately 300 µL of this dilute DAPI staining solution to the coverslip preparation, making certain that the cells are completely covered.
5. Incubate for 1–5 minutes.
6. Rinse the sample several times in PBS. Drain excess buffer from the coverslip and mount. You may choose to use a mounting medium with an antifade reagent.
7. View the sample using a fluorescence microscope with an appropriate excitation source and emission filter. The excitation maximum for DAPI bound to dsDNA is 358 nm, and the emission maximum is 461 nm.

Staining Suspension Cells for Flow Cytometry or Microscopy:

Sample Preparation: if fixing cells, use the fixation protocol appropriate for your sample, or use the following protocol.

1. Collect a cell suspension of 2×10⁵ - 1×10⁶ cells.
2. Pellet the cells by centrifugation and discard the supernatant.
3. Tap the tube to resuspend the pellet in the residual liquid and add 1 mL of PBS at room temperature.
4. Transfer the full volume of resuspended cells to 4 mL of fixative (0.5% Paraformaldehyde) by pipetting the cell suspension slowly into the fixative while vortexing at top speed. Leave the cells in fixative for 5-15 minutes.
5. Pellet the cells by centrifugation and discard the fixative.
6. Tap the tube to loosen the pellet and add 5 mL of PBS at room temperature. Allow the cells to rehydrate for 15 minutes.

Staining and Analysis:

1. 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.
2. Centrifuge the cell suspension (from Sample Preparation Step 6) and discard the supernatant. Tap to loosen the pellet and add 1 mL of 3 µM DAPI.
3. Incubate for 15 minutes at room temperature.
4. Analyze samples by flow cytometry; centrifugation is not necessary. DAPI may be used in flow cytometry systems utilizing UV excitation sources. Its emission maximum is 461 nm.
5. If the cells are to be viewed by fluorescence microscopy, centrifuge the sample, remove the supernatant, and resuspend cells in fresh staining buffer. Apply a drop of the suspension to a microscope slide, apply a coverslip and view. The excitation maximum for DAPI bound to dsDNA is 358 nm, and the emission maximum is 461 nm.