How do you use a Hoechst stain?
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.
What is the Hoechst method?
Hoechst 33342 is used for specifically staining the nuclei of living or fixed cells and tissues. This stain is commonly used in combination with 5-bromo-2′-deoxyuridine (BrdU) labeling to distinguish the compact chromatin of apoptotic nuclei, to identify replicating cells and to sort cells based on their DNA content.
How long does DAPI staining last?
six months
For long-term storage the stock solution can be aliquoted and stored at =–20°C. For short-term storage the solution can be kept at 2–6°C, protected from light. When handled properly, DAPI solutions are stable for at least six months.
Why is Hoechst better than DAPI?
Hoechst dyes are less toxic than DAPI, which ensures a higher viability of stained cells. The additional ethyl group of the Hoechst dyes renders them more cell-permeable. There are nuclei staining dyes that allow for viability of cells after staining.
Does Hoechst bind to RNA?
3.3 Hoechst 33258 fluoresces only about half as much when it binds to single-stranded genomic DNA compared to when it binds to double- stranded genomic DNA. 3.6 RNA does not interfere significantly with the DNA assay because Hoechst 33258 does not normally bind to RNA.
How much should I add to DAPI?
Preparing solutions
- Add 2 mL of 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.
- Add 2.1 µL of the 14.3 mM DAPI stock solution to 100 µL PBS to make a 300 µM DAPI intermediate dilution.
What do you dilute DAPI in?
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. Rinse samples once in PBS and then prepare for imaging. Examine immediately using appropriate excitation wavelength.