Testing Data
Hoechst 33258 Reagent
Hoechst 33258 Staining Dye Solution
Applications
Immunofluorescence, Flow Cytometry, Functional Assay
Synonyms
Hoechst 33258; Hoechst 33258 Staining Dye Solution; Hoechst 33258 reagent
Form/Format
Liquid
Concentration
1ug/ml (varies by lot)
Applicable Applications for Hoechst 33258 reagent
Immunofluorescence (IF), Flow Cytometry (FC/FACS)
Application Notes
Fluorescent bioimaging; Immunofluorescence assays, flow cytometry; labeling of DNA in cell cultures; cell death detection
Molecular formula
C25H24N6O 3(HCl) x(H2O)
Excitation DAPI
346nm
Emission DAPI
460nm
Excitation DAPI-DNA complexes
352nm
Emission DAPI-DNA complexes
461nm
Usage and Handling
In vitro laboratory use only. Not for any clinical, therapeutic, or test use in humans or animals.
It is recommended to complete the test on the same day after dyeing to avoid fluorescence quenching. Live cells or tissues should be observed immediately after staining.
Antifade Mounting Medium could be used to prevent rapid photobleaching of fluorescence within spectrum range.
Hoechst 33258 should be handled with care.
It is recommended to complete the test on the same day after dyeing to avoid fluorescence quenching. Live cells or tissues should be observed immediately after staining.
Antifade Mounting Medium could be used to prevent rapid photobleaching of fluorescence within spectrum range.
Hoechst 33258 should be handled with care.
Protocol
Fixed cells and tissues:
1. Wash appropriately to remove fixative. If necessary, immunofluorescent staining can be performed first, then perform the Hoechst 33258 staining. If there is no other staining, perform Hoechst 33258 staining directly.
Adherent cells or tissue slices: add a sufficient volume of Hoechst 33258 Staining Dye Solution to completely cover the sample.
Suspension cells: add Hoechst 33258 Staining Dye Solution to suspend cell pellets at 3: 1. Vortex to obtain an even suspension.
2. Incubate for 3-5 minutes at RT(room temperature).
3. Remove the stain solution.
4. Wash with TBST, PBS or physiological saline for 2-3 times, 3-5 minutes each.
5. Observe the sample under fluorescence microscope (excitation wavelength: 360nm, emission wavelength: 460nm).
When cell apoptosis happens, the nucleus of the apoptotic cell will be densely stained, or it will be fragmented and densely stained.
Live cells and tissues:
1. Add a sufficient volume of Hoechst 33258 Staining Dye Solution to completely cover the sample. For 6 well plate, add 1ml Hoechst 33258 Staining Dye Solution into each well. For 96 well plate, add 100ul Hoechst 33258 Staining Dye Solution into each well.
2. Incubate for 20-30 minutes at an appropriate temperature for cells.
3. Remove the stain solution.
4. Wash with PBS or culture medium for 2-3 times, 3-5 minutes each.
5. Observe the sample under fluorescence microscope (excitation wavelength: 360nm, emission wavelength: 460nm).
When cell apoptosis happens, the nucleus of the apoptotic cell will be densely stained, or it will be fragmented and densely stained.
1. Wash appropriately to remove fixative. If necessary, immunofluorescent staining can be performed first, then perform the Hoechst 33258 staining. If there is no other staining, perform Hoechst 33258 staining directly.
Adherent cells or tissue slices: add a sufficient volume of Hoechst 33258 Staining Dye Solution to completely cover the sample.
Suspension cells: add Hoechst 33258 Staining Dye Solution to suspend cell pellets at 3: 1. Vortex to obtain an even suspension.
2. Incubate for 3-5 minutes at RT(room temperature).
3. Remove the stain solution.
4. Wash with TBST, PBS or physiological saline for 2-3 times, 3-5 minutes each.
5. Observe the sample under fluorescence microscope (excitation wavelength: 360nm, emission wavelength: 460nm).
When cell apoptosis happens, the nucleus of the apoptotic cell will be densely stained, or it will be fragmented and densely stained.
Live cells and tissues:
1. Add a sufficient volume of Hoechst 33258 Staining Dye Solution to completely cover the sample. For 6 well plate, add 1ml Hoechst 33258 Staining Dye Solution into each well. For 96 well plate, add 100ul Hoechst 33258 Staining Dye Solution into each well.
2. Incubate for 20-30 minutes at an appropriate temperature for cells.
3. Remove the stain solution.
4. Wash with PBS or culture medium for 2-3 times, 3-5 minutes each.
5. Observe the sample under fluorescence microscope (excitation wavelength: 360nm, emission wavelength: 460nm).
When cell apoptosis happens, the nucleus of the apoptotic cell will be densely stained, or it will be fragmented and densely stained.
Preparation and Storage
Upon receipt store at -20 degree C, protect from light. When stored as directed, product should be stable for one year.
Testing Data
Testing Data
Related Product Information for Hoechst 33258 reagent
Description: Hoechst 33258 Staining Dye Solution is a fluorescent stain for labeling DNA in fluorescence microscopy. This product may be used in fluorescence microscopy, microplate, cuvette, and flow cytometry applications. Hoechst 33258 may be used on live or fixed cells and is also applicable for cell cycle analysis and monitoring DNA condensation by flow cytometry.
Background: The Hoechst stains are a family of fluorescent stains for labeling DNA in fluorescence microscopy. Because these fluorescent stains label DNA, they are also commonly used to visualize nuclei and mitochondria. Two of these closely related bis-benzimides are commonly used: Hoechst 33258 and Hoechst 33258. Both dyes are excited by ultraviolet light at around 350 nm, and both emit blue/cyan fluorescence light with an emission maximum at 461 nm. The Hoechst stains may be used on live or fixed cells, and are often used as a substitute for another nucleic acid stain, DAPI. The key difference between them is that the additional ethyl group of Hoechst 33258 makes it more lipophilic, and thus more readily to cross intact cell membranes. In some applications, Hoechst 33258 is significantly less permeant than Hoechst 33258.
Background: The Hoechst stains are a family of fluorescent stains for labeling DNA in fluorescence microscopy. Because these fluorescent stains label DNA, they are also commonly used to visualize nuclei and mitochondria. Two of these closely related bis-benzimides are commonly used: Hoechst 33258 and Hoechst 33258. Both dyes are excited by ultraviolet light at around 350 nm, and both emit blue/cyan fluorescence light with an emission maximum at 461 nm. The Hoechst stains may be used on live or fixed cells, and are often used as a substitute for another nucleic acid stain, DAPI. The key difference between them is that the additional ethyl group of Hoechst 33258 makes it more lipophilic, and thus more readily to cross intact cell membranes. In some applications, Hoechst 33258 is significantly less permeant than Hoechst 33258.
Product Categories/Family for Hoechst 33258 reagent
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Product Notes
The Hoechst 33258 (Catalog #AAA1753887) is a Reagent and is intended for research purposes only. The product is available for immediate purchase. AAA Biotech's Hoechst 33258 can be used in a range of immunoassay formats including, but not limited to, Immunofluorescence (IF), Flow Cytometry (FC/FACS). Fluorescent bioimaging; Immunofluorescence assays, flow cytometry; labeling of DNA in cell cultures; cell death detection. Researchers should empirically determine the suitability of the Hoechst 33258 for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "Hoechst 33258, Reagent" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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