Br-DAPI targets DNA, specifically binding to the A/T-rich regions in the minor groove of double-stranded DNA. The compound's binding is highly selective for A/T base pairs, with minimal binding to G/C-rich sequences. The bromine substitution may enhance the compound's photophysical properties and enable photodynamic activity upon light activation. As a DNA-binding dye, Br-DAPI's primary target is the DNA molecule itself, making it useful for nuclear staining in various imaging applications. Its cell-permeability allows it to reach intracellular DNA in live cells.

Usage instructions 1. Creating the functioning DAPI solution 1.1 Getting the stock solution ready To make a 1 mg/mL stock solution, dilute 1 milligram of DAPI with 1 mL of double-distilled water. It is advised to aliquot the DAPI storage solution and keep it in the dark at -20°C or -80°C. 1.2 Making a functional solution To make a 1-10 μg/mL DAPI working solution, use either PBS dilution storage solution or hot serum-free cell culture media. Note: Before using the DAPI working fluid, please make sure that its concentration is appropriate for the current circumstances. 2. Staining of suspended cells 2.1 Centrifuge the cells to collect them, then add PBS and wash twice for five minutes each time. There are 1×106 cells per milliliter. 2.2 Add 1 milliliter of DAPI working solution, then let it sit at room temperature for three to ten minutes. 2.3 After centrifuging for three to four minutes at 400 g, remove the supernatant. 2.4 Add PBS and give the cells two 5-minute washings. 2.5 Re-suspend the cells in 1 mL of PBS or serum-free media, and use a flow cytometer or fluorescence microscope to observe. 3. Adherent cell staining 3.1 Use sterile coverslips to culture adherent cells. 3.2 Aspirate the extra culture medium after removing the coverslip from the medium. 3.3 Add 100 μL of the dye working solution, give the cells a gentle shake to cover them completely, and then let them sit for three to ten minutes. 3.4 Aspirate the dye working solution, wash with culture medium two or three times for five minutes each time, and use a flow cytometer or fluorescence microscope to monitor. Storage conditions: -20°C, one year of light protection. Notes: 1. Please modify the concentration of the DAPI working solution and the incubation period in accordance with the actual circumstances. 2. Professionals may only use this product for scientific study; it may not be utilized for clinical diagnosis or treatment, nor may it be included into food or medication. 3. Please wear a lab coat and disposable gloves for your own health and safety.

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In vitro studies demonstrate that Br-DAPI is a potent DNA-binding dye with enhanced photophysical properties compared to DAPI. The compound binds to A/T base pairs in the minor groove of double-stranded DNA with high affinity, resulting in significant fluorescence enhancement upon binding. Its fluorescence excitation and emission maxima are similar to DAPI, making it compatible with standard DAPI filter sets. The bromine substitution may improve photostability and enable photodynamic activity. The compound's in vitro activity is assessed by measuring fluorescence intensity upon DNA binding, DNA staining efficiency in fixed and live cells, and photodynamic activity upon light activation.

Cellular assays for Br-DAPI are conducted to evaluate its DNA staining efficiency and photodynamic activity. Cells are incubated with the compound at appropriate concentrations (typically 0.1-10 microg/mL) for 10-30 minutes at 37degC. For live cell imaging, the compound is added to culture medium and cells are incubated under standard conditions. For fixed cell imaging, cells are fixed with paraformaldehyde or methanol prior to staining. Stained cells are washed and imaged using fluorescence microscopy with DAPI filter sets. Fluorescence intensity is quantified to assess DNA content or cell cycle distribution. Photodynamic activity is assessed by irradiating stained cells with light and measuring cell viability or DNA damage.

In vivo applications of Br-DAPI are primarily related to its use as a DNA stain rather than a therapeutic agent. The compound may be used in animal models for labeling cells or tissues ex vivo, though its in vivo use is limited due to potential toxicity and rapid clearance. When used in vivo, the compound is typically administered locally for tissue staining rather than systemically. Its utility in vivo is primarily for research applications such as tracking cell populations or assessing tissue DNA content. The compound is not intended for therapeutic use.

In vitro enzyme/receptor binding assays are not applicable for Br-DAPI as it is a DNA-binding dye rather than an enzyme inhibitor. Instead, DNA binding assays are used to characterize its interaction with DNA. These assays typically involve incubating the compound with purified DNA (e.g., calf thymus DNA) or synthetic oligonucleotides containing A/T-rich sequences, and measuring fluorescence enhancement upon binding. Binding affinity is determined by fluorescence titration, and binding stoichiometry is assessed by Scatchard analysis. The compound's specificity for A/T base pairs is confirmed by comparing binding to A/T-rich versus G/C-rich DNA sequences.

Cellular assays for Br-DAPI are the primary method for evaluating its utility as a DNA stain. Cells are cultured in appropriate media and treated with the compound at concentrations typically ranging from 0.1-10 microg/mL for 10-30 minutes. For live cell imaging, the compound is added directly to the culture medium. For fixed cell imaging, cells are fixed prior to staining. Stained cells are washed to remove unbound dye and imaged using fluorescence microscopy with DAPI filter sets. Fluorescence intensity is quantified using image analysis software. Cell cycle analysis is performed by flow cytometry to assess DNA content. Cytotoxicity is assessed using MTT or similar assays.

In vivo animal studies with Br-DAPI are limited, as the compound is primarily used as a research tool for DNA staining rather than as a therapeutic agent. When used in vivo, the compound may be administered to animals for tissue staining or cell labeling ex vivo. For example, tumor-bearing mice may be injected with the compound to label tumor cells for imaging studies, though such applications are less common. The compound's rapid clearance and potential toxicity limit its in vivo utility. Detailed in vivo study protocols are not extensively reported in the literature.

Pharmacokinetic properties of Br-DAPI are characteristic of small molecule DNA-binding dyes. The compound has a molecular weight of 356.22 g/mol and a molecular formula of C1₆H14BrN₅. It is water-soluble, facilitating its use in aqueous biological systems. The compound is cell-permeable, allowing it to enter live cells. It is typically stored at 4degC or -80degC, protected from light. Detailed PK parameters such as half-life and bioavailability are not reported, as the compound is not used as a therapeutic agent.

[1]. DAPI: a DNA-specific fluorescent probe. Biotech Histochem. 1995 Sep;70(5):220-33.

Toxicological data for Br-DAPI are limited, as the compound is primarily used as a research tool for DNA staining rather than as a therapeutic agent. The compound is not intended for human therapeutic use and is supplied for research purposes only. In cell-based assays, the compound is generally well-tolerated at concentrations used for DNA staining (0.1-10 microg/mL), though higher concentrations may cause cytotoxicity. The compound's DNA-binding properties suggest potential genotoxicity if it causes DNA damage or interferes with DNA replication. Standard safety precautions should be followed when handling this compound.

C16H14BRN5

356.219861507416

355.04

2387906-44-5

162642091

Light yellow to yellow solid powder

2.3

5

2

3

22

446

0

BrC1=C(C2C=CC(C(=N)N)=CC=2)NC2C=C(C(=N)N)C=CC=21

GSBXNZFEZFIMCS-UHFFFAOYSA-N

InChI=1S/C16H14BrN5/c17-13-11-6-5-10(16(20)21)7-12(11)22-14(13)8-1-3-9(4-2-8)15(18)19/h1-7,22H,(H3,18,19)(H3,20,21)

3-bromo-2-(4-carbamimidoylphenyl)-1H-indole-6-carboximidamide

Br-DAPI

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)