| Size | Price | Stock | Qty |
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| 50mg |
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| 100mg |
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| 250mg |
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| 500mg |
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| 1g |
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| Other Sizes |
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Purity: ≥98%
| Targets |
Probe to detect intracellular ROS
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|---|---|
| ln Vitro |
Use guide (this is our suggested plan; it should be adjusted to meet your individual needs; it is merely a guide).
1. To prepare a 10 mM extraction solution, dissolve H2DCFDA in DMSO. Then, extract again before to usage. 2. After staining the cells for 30 minutes at 37°C in the dark with PBS solution containing 5 μM H2DCFDA and 0.05% trypsin-EDTA solution, the cells were suspended in new culture medium and subjected to an instant flow cytometry analysis. 3. If required, employ the H2DCFDA probe in conjunction with the ROS-insensitive fluorescein dye DCFDA modifier as an improved control. The staining process is identical to that of H2DCFDA [1]. |
| ln Vivo |
Results: Our studies identify that; (1) frequencies and absolute numbers of DCs in the spleen and BM are altered on day 3 and day 7 after TBI; (2) surface expression of key molecules involved in antigen presentation of DCs were affected on day 3 and day 7 after TBI; (3) distribution and functions of tissue-specific DC subsets of both circulatory and lymphatic systems were imbalanced following TBI; (4) early differentiation program of DCs, especially the commitment of hematopoietic stem cells to common DC progenitors (CDPs), were deregulated after TBI; and (5) intracellular ROS levels were reduced in DC progenitors and differentiated DCs on day 3 and day 7 after TBI.[2]
Conclusions: Our data demonstrate, for the first time, that TBI affects the distribution pattern of DCs and induces an imbalance among DC subsets in both lymphoid and non-lymphoid organs. In addition, the current study demonstrates that TBI results in reduced levels of ROS in DCs on day 3 and day 7 after TBI, which may explain altered DC differentiation paradigm following TBI. A deeper understanding on the molecular mechanisms that contribute to DC defects following TBI would be essential and beneficial in treating infections in patients with acute central nervous system (CNS) injuries, such as TBI, stroke and spinal cord injury.[2] |
| Enzyme Assay |
Staining Example 1:
H2DCFDA (DCFH-DA) is a ROS production probe with green fluorescence. 1. Remove the cell culture medium and replaced by fresh medium containing H2DCFDA and incubated with cultured cells for 30 min. 2. Wash cells with PBS for three times. 3. Use a confocal laser scanning microscopy to detect the intracellular ROS generation. Staining Example 2: 1. Testing cells are seeded into a 6-well microplate at a density of 105/well and cultured overnight at 37°C in 5% CO2. Fresh medium with 50 μg/mL of different materials is added to each well and cultured for 16 h. 2. Wash cells with PBS and stain cells with H2DCFDA (20 μM; 1 h; 37°C; dark). 3. Cells are trypsinized, washed, resuspended in PBS (0.2 mL), analyzed by a flow cytometer and use a confocal laser scanning microscopy (FV1000, Olympus, Tokyo, Japan) for image. Staining Example 3: 1. Wash cultured cells with PBS for three times. 2. Treat testing cells with H2DCFDA in culture medium for 0.5 h in a cell incubator. 3. Use a confocal laser scanning microscopy (Olympus, FV3000) for image. Cells are analyzed on a BD LSRI Fortessa flow cytometer analyzer to quantify results. |
| Cell Assay |
Guidelines (The following is our recommended method, which only provides guidelines and should be modified according to your specific needs).
1.1 Preparation of stock solution Prepare 5 mM of H2DCFDA stock solution with anhydrous DMSO. Note: H2DCFDA stock solution is recommended to be stored in the dark at -20 ℃ or -80 ℃ after aliquot. 1.2 Preparation of working solution Dilute the stock solution with preheated serum-free cell culture medium or PBS to prepare 1-10 μ M H2DCFDA working solution. Note: Please adjust the concentration of H2DCFDA working solution according to your specific needs, and prepare it as needed. 2. Cell staining (suspended cells) 2.1 Centrifuge and collect cells, wash twice with PBS for 5 minutes each time. The cell density is 1 × 106/mL. 2.2 Add 1 mL of dye working solution and incubate at room temperature for 5-30 minutes. 2.3 400 g, centrifuge for 3-4 minutes, discard the supernatant. 2.4 Add PBS to wash cells twice, each time for 5 minutes. After resuspending cells in 1 mL serum-free medium or PBS, observe using a fluorescence microscope or flow cytometry. 3. Cell staining (adherent cells) 3.1 Cultivate adherent cells on sterile cover slips. 3.2 Remove the cover glass from the culture medium and remove excess culture medium. 3.3 Add 100 μ L of dye working solution, gently shake to completely cover the cells, and incubate for 5-30 minutes. 3.4 Remove the dye working solution, wash 2-3 times with culture medium for 5 minutes each time, and observe using a fluorescence microscope or flow cytometer. Note: If flow cytometry detection is required, the cells need to be digested and resuspended with trypsin before staining. |
| Animal Protocol |
Long bones, spleen, peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), liver, lungs, skin and blood were collected from mice with either moderate-level cortical impact (CCI) or sham on day 1, day 3 or day 7 after TBI. Bone marrow cells were isolated from the tibias and femurs of hind limb through flushing. Tissues were digested with Collagenase-D and DNase I. Skin biopsies were digested in the presence of liberase + DNase I. Single cell suspensions were made, red blood cells were lysed with Ammonium chloride (Stem Cell Technology) and subsequently filtered using a 70 μM nylon mesh. DC subsets of the tissues and DC progenitors of the BM were identified through 10-color flow cytometry-based immunophenotyping studies. Intracellular reactive oxygen species (ROS) were identified through H2DCFDA staining.[2]
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| References |
[1]. Redox environment in stem and differentiated cells: A quantitative approach. Redox Biol. 2017 Aug;12:758-769.
[2]. J Neuroinflammation. 2022 Oct 1;19(1):238. |
| Additional Infomation |
See also: Diacetyldichlorofluorescein (annotation moved to).
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| Molecular Formula |
C24H16CL2O7
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|---|---|
| Molecular Weight |
487.2856
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| Exact Mass |
486.027
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| CAS # |
4091-99-0
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| Related CAS # |
4091-99-0;
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| PubChem CID |
77718
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| Appearance |
Typically exists as white to off-white solids at room temperature
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
593.5±50.0 °C at 760 mmHg
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| Melting Point |
209-210ºC(lit.)
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| Flash Point |
312.7±30.1 °C
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| Vapour Pressure |
0.0±1.8 mmHg at 25°C
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| Index of Refraction |
1.635
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| LogP |
4.62
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
33
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| Complexity |
717
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=C(C([H])=C2C(=C1[H])C([H])(C1=C([H])C([H])=C([H])C([H])=C1C(=O)O[H])C1=C([H])C(=C(C([H])=C1O2)OC(C([H])([H])[H])=O)Cl)OC(C([H])([H])[H])=O
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| InChi Key |
PXEZTIWVRVSYOK-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H16Cl2O7/c1-11(27)31-21-9-19-15(7-17(21)25)23(13-5-3-4-6-14(13)24(29)30)16-8-18(26)22(32-12(2)28)10-20(16)33-19/h3-10,23H,1-2H3,(H,29,30)
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| Chemical Name |
2-(3,6-diacetyloxy-2,7-dichloro-9H-xanthen-9-yl)benzoic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~125 mg/mL (~256.52 mM)
Ethanol : ~20 mg/mL (~41.04 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (4.27 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0522 mL | 10.2608 mL | 20.5217 mL | |
| 5 mM | 0.4104 mL | 2.0522 mL | 4.1043 mL | |
| 10 mM | 0.2052 mL | 1.0261 mL | 2.0522 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Products are for research use only; We do not sell to patients
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