| Size | Price | Stock | Qty |
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| 50mg |
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| 100mg |
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| 250mg |
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| Other Sizes |
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| Targets |
Fluorescent Dye
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|---|---|
| ln Vitro |
In situ bone osteoclast numbers[1]
Quantitation of osteoclasts in demineralized bone sections was based on previously described procedures. Fixed, demineralized, and paraffin embedded tibiae were sectioned and subjected to staining for tartrate resistant acid phosphatase (TRAP) with 0.1 mg/ml naphthol AS-MX phosphate and 0.6 mg/ml fast red violet LB salt in 0.1 M sodium acetate buffer, pH 5.0, containing 50mM sodium tartrate. Osteoclasts were identified as TRAP positive cells on the trabecular bone surfaces. The numbers of osteoclasts on the sections were counted and the fractions of bone surface occupied by osteoclasts were measured in the proximal tibia trabeculae using a color camera microscopy imaging system and the software of ImageJ (1.35s, NIH). Osteoclast potential[1] The bone marrow cells were cultured (1.0 × 105 cells/0.5ml per well in a 48-well plate) for 6 days in α-MEM containing 10% FBS, 100U/ml penicillin, and 100μg/ml streptomycin. Cultures were fed every 3 days with medium containing rmM-CSF (20 ng/ml), rhsRANKL (60 ng/ml) and maintained at 37°C in a humidified atmosphere of 5% CO2. On day 6, cells were fixed in 10% formalin and stained for TRAP with 0.1 mg/ml naphthol AS-MX phosphate and 0.6 mg/ml fast red violet LB salt in 0.1 M sodium acetate buffer, pH 5.0, containing 50mM sodium tartrate. TRAP-positive cells with three or more nuclei were counted using phase-contrast microscopy. |
| Cell Assay |
Cell viability and differentiation assay[2]
The viability of the cryopreserved cells was assayed using a NucleoCounter, an instrument for counting mammalian cells, which employs a fluorescence microscope adapted to a relatively low optical magnification. The cell samples were analyzed on the NucleoCounter before and after treatment with a lysis buffer, giving an estimate of nonviable and total cells. To monitor osteoblastic differentiation, the cryopreserved cells were thawed in α-MEM containing 15% FBS. They were then seeded and cultured at a density of 1 × 104 cells/cm2 in a twelve-well culture plate in the medium supplemented with 10 mM β-glycerophosphate disodium salt, 0.07 mM L-ascorbic acid phosphate magnesium salt n-hydrate and 0.1 mM dexamethasone for two weeks. The cultures without dexamethasone were used as a negative control. Differentiated osteoblasts were biochemically analyzed by the determination of calcium and ALP activity staining. Calcium deposition (in vitro bone formation) was evaluated by the method we previously reported). Briefly, 1 µg/mL calcein was added to the medium during the culture period and the fluorescence of the incorporated calcein in the extracellular regions of the cells was observed by using a fluorescent microscope. The medium containing the calcein was removed and washed with PBS prior to observation. For alkaline phosphatase (ALP) activity staining, the cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min at 4°C. The fixed cells were then soaked in 0.1% naphthol AS-MX phosphate and 0.1% fast red violet LB salt in 56 mM 2-amino-2-methyl-1, 3-propanediol for 10 min at room temperature. Following a washing step with PBS, the active ALP cells were observed by microscopy[2]. |
| References |
[1]. Yan D, et al. Genetic background influences fluoride's effects on osteoclastogenesis. Bone. 2007 Dec;41(6):1036-44.
[2]. Kotobuki N, et al. Cultured autologous human cells for hard tissue regeneration: preparation and characterization of mesenchymal stem cells from bone marrow. Artif Organs. 2004 Jan;28(1):33-9. |
| Molecular Formula |
C14H11CLN3O.1/2ZNCL2
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|---|---|
| Molecular Weight |
340.86
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| Related CAS # |
Fast Red Violet LB;32348-81-5
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| Appearance |
Off-white to yellow solid
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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 |
| 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 :~62.5 mg/mL (~183.36 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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 (6.10 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9338 mL | 14.6688 mL | 29.3376 mL | |
| 5 mM | 0.5868 mL | 2.9338 mL | 5.8675 mL | |
| 10 mM | 0.2934 mL | 1.4669 mL | 2.9338 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.
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