DAPI dihydrochloride

Biological Activity

DAPI dihydrochloride is a blue fluorescent nucleic acid stain, preferentially binds dsDNA and associates with the minor groove AT clusters. Association of DAPI with dsDNA increases fluorescence approximately 20-fold, with an emission maximum of 460 nm. The increase in fluorescence is believed to be due to displacement of water molecules from the minor groove and DAPI. DAPI can also bind RNA and emits at a longer wavelength of 500 nm, with only a 20% increase in fluorescence.

Protocols for Counterstaining with DAPI:

Immunofluorescence Staining
Note: Cells may be fixed by method of choice, such as with 4% paraformaldehyde. DAPI staining is done after staining for other markers. Fixation and permeabilization of cells is not necessary to counterstain with DAPI.

1. Fix cells using method of choice.
2. Incubate the cells with phosphate buffered saline (PBS) for 15 minutes.
3. Dilute the DAPI solution (we recommend to 300 nM) with PBS. Remove excess PBS from the slide and cover with DAPI solution, making sure the cells are completely covered.
4. Incubate for up to 5 minutes. Rinse the slide several times to remove all free DAPI. It is recommended to use a mounting medium with antifade reagent to reduce fluorescence quenching. The slide is ready to view under the microscope with appropriate filters.


Nuclear Staining for Flow Cytometry
Note: Cells may be fixed with 4% paraformadehyde or absolute ethanol.

1. Fix cells with absolute ethanol or 4% paraformaldehyde.
2. Centrifuge, discard the supernatant, and add 5 ml of PBS, allowing cells to rehydrate for 15 minutes.
3. Dilute the DAPI to 3 µM (recommended) in staining buffer.
4. Centrifuge cell suspension from step 3 above, discard supernatant, and add 1 ml of DAPI solution, then incubate at room temperature for 15 minutes.
5. Analyze by flow cytometry using an instrument with appropriate lasers.

Product Citations

• 

  Adv Mater. 2023 Feb 22;e2211138.

  Genetically Programmable Vesicles for Enhancing CAR-T Therapy Against Solid Tumors
  DAPI dihydrochloride purchased from AbMole

• 

  Nucleic Acids Res. 2022 Aug 12;50(14):7938-7958.

  HDAC1 and PRC2 mediate combinatorial control in SPI1/PU.1-dependent gene repression in murine erythroleukaemia
  DAPI dihydrochloride purchased from AbMole

• 

  Oncol Lett. 2022 May;23(5):163.

  AK4P1 is a cancer-promoting pseudogene in pancreatic adenocarcinoma cells whose transcripts can be transmitted by exosomes
  DAPI dihydrochloride purchased from AbMole

• 

  Appl Biochem Biotechnol Appl Biochem Biotechnol. 2022 Jul 1.

  Apatinib Through Activating the RhoA/ROCK Signaling Pathway to Cause Dysfunction of Vascular Smooth Muscle CellsMuscle Cells
  DAPI dihydrochloride purchased from AbMole

• 

  J Nanobiotechnology. 2021 Nov 25;19(1):391.

  Nanovesicles derived from bispecific CAR-T cells targeting the spike protein of SARS-CoV-2 for treating COVID-19
  DAPI dihydrochloride purchased from AbMole

• 

  Ann Biomed Eng. 2021 Jun;49(6):1551-1560.

  Comparison of in Vascular Bioreactors and In Vivo Models of Degradation and Cellular Response of Mg-Zn-Mn Stents
  DAPI dihydrochloride purchased from AbMole

• 

  Reprod Sci. 2021 Jun 2.

  LncRNA HOXA-AS2 Activates the Notch Pathway to Promote Cervical Cancer Cell Proliferation and Migration
  DAPI dihydrochloride purchased from AbMole

• 

  Theranostics. 2020 Apr 27;10(13):5671-5686.

  SUMO1 modification of methyltransferase-like 3 promotes tumor progression via regulating Snail mRNA homeostasis in hepatocellular carcinoma
  DAPI dihydrochloride purchased from AbMole

• 

  Aging (Albany NY). 2020 Jun 9;12(11):11025-11041.

  LncRNA ADAMTS9-AS2 inhibits gastric cancer (GC) development and sensitizes chemoresistant GC cells to cisplatin by regulating miR-223-3p/NLRP3 axis
  DAPI dihydrochloride purchased from AbMole

• 

  Life Sci. 2020 Sep 15;257:118021.

  TRIB3 destabilizes tumor suppressor PPARα expression through ubiquitin-mediated proteasome degradation in acute myeloid leukemia
  DAPI dihydrochloride purchased from AbMole

• 

  Oncology. 2020 Jul 20.

  5-Fluorouracil enhances the chemosensitivity of gastric cancer to TRAIL via inhibition of the MAPK pathway
  DAPI dihydrochloride purchased from AbMole

• 

  Anat Rec (Hoboken). 2020 Jan 21.

  Resibufogenin inhibited colorectal cancer cell growth and tumorigenesis through triggering ferroptosis and ROS production mediated by GPX4 inactivation
  DAPI dihydrochloride purchased from AbMole

Chemical Information

Molecular Weight	350.25
Formula	C16H17Cl2N5
CAS Number	28718-90-3
Solubility (25°C)	Water 5 mg/mL (Need ultrasonic)
Storage	-20°C, protect from light, sealed

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Qunxiong Wu, et al. Reprod Sci. LncRNA HOXA-AS2 Activates the Notch Pathway to Promote Cervical Cancer Cell Proliferation and Migration