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Hoechst 33258, pentahydrate (bis-benzimide), 100 mg - FAQs

Hoechst 33258, pentahydrate (bis-benzimide), 100 mg - FAQs

View additional product information for DAPI and Hoechst Nucleic Acid Stains - FAQs (H21491, H21492, H3569, H1398, D3571, H1399, D21490, D1306, H3570)

6 product FAQs found

Can I use Hoechst dyes to do both DNA quantitation and cell number counting in plates?

Yes, this can be done in a 96-well plate-based format. Hoechst dyes will label all cells, live or dead (or even fixed). This requires the use of a standard curve of known cell numbers (up to 100,000 cells per well) and a standard curve using purified DNA, , up to 10 µg/mL. We offer a kit for this: FluoReporter Blue Fluorometric dsDNA Quantitation Kit, Cat. No. F2962.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Is DAPI a good live-cell nuclear label?

DAPI is considered a semi-permeant/impermeant nucleic acid stain. Staining of nucleic is dependent upon the cell line in its performance. Some cell lines will label with DAPI, others not at all, and others label inconsistenly. Instead, we recommend using either Hoechst 33342 or Hoechst 33258, which have the same wavelength and binding mode as DAPI (at the A-T minor groove) but are readily cell-permeant.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I want to label the nuclei of live cells and track them over time. Can I use DAPI for this?

We do not recommend doing this. DAPI is considered to be a semi-permeant/impermeant nucleic acid stain. DAPI staining of live cells may be inconsistent. It is best used as a counterstain for fixed samples. Other cell permeable nucleic acid stains, such as Hoechst or the SYTO dyes may affect cellular function.

For mammalian cells, we recommend using the CellLight Nucleus transduction reagents, available in CFP, GFP and RFP. With these reagents, the cells are transduced overnight in a single labeling step and the next day the nuclei will fluoresce. The label may be retained for 3-5 days and should not affect cell function. Cytoplasmic cell tracking dyes such as the CellTracker dyes may also be used.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Can I use a nuclear counterstain with Invitrogen CellEvent Caspase Detection Reagents?

Yes, but add the nuclear counterstain after the Invitrogen CellEvent Caspase reagent signal has developed. For kinetic or continuous-culture experiments, add a Hoechst nuclear stain at the endpoint to reduce potential effects on normal cell physiology. Nuclear counterstain dyes may compete for nucleic acid binding, so optimize the combination.

I have a mix of live bacteria and platelet cells, and I need to be able to separate out the bacteria. Do you have a suggestion?

Platelet cells don't have DNA, while bacteria do. Therefore, a cell-permeant, DNA-selective dye would preferentially stain the bacteria with limited staining of the platelets. We recommend using Hoechst 33342 dye.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

DAPI and Hoechst dyes are quite similar to each other. Why would I choose one over the other?

DAPI is a very common blue-fluorescent dye for nuclear counterstaining and gives very bright labeling on nuclei in fixed and permeabilized cells and tissues. However, it is considered to be a semi-permeant to impermeant stain and provides inconsistent staining of live cells. Hoechst 33342 dye is cell-permeant and stains with the same binding mechanism and fluorescent color; it is preferred for live-cell imaging and is just as good as DAPI for fixed cell labeling.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.