| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
Caspase-8
|
|---|---|
| ln Vitro |
Marked caspase-8 activation was demonstrated in a minority of isolated pancreatic acinar cells by using a fluorescent probe-linked caspase-8 substrate (Z-IETD-R110). Menadione (30 μM) induced a significant increase (P < 0.00018) in the fluorescence of 15 ± 2% of acinar cells (Fig. 1B). Apoptosis-positive cells displayed a high increase in fluorescence (∼10.9 ± 0.8-fold) compared with control cells (Fig. 2A). Fluorescence of the caspase-8 substrate increased within 30 min after application of menadione (Fig. 2B). These data show that caspase-8 was activated in response to the menadione-induced oxidative stress. Confocal-microscopy images (Fig. 2C) show homogeneous distribution of fluorescence throughout the cell, suggesting predominantly cytosolic localization of activated caspase-8. [1]
|
| Cell Assay |
Caspase activation. [1]
Isolated pancreatic acinar cells were washed and suspended in calcium-free buffer solution (140 mM NaCl, 1.13 mM MgCl2, 4.7 mM KCl, 10 mM glucose, 0.1 M EDTA, and 10 mM HEPES, pH 7.2). Cells were then loaded with fluorescent indicator-linked substrates for activated caspase-2 (10 μM Z-VDAD-R110), caspase-8 (10 μM Z-IETD-R110), caspase-9 (10 μM Z-LEHD-R110), or general caspases (10 μM R110-aspartic acid amide) at room temperature for 20 min or for caspase-10 (50 μM AEVD-AFC) or caspase-12 (50 μM ATAD-AFC) at 37°C for 1 h. Caspase substrates (except for the general substrate) used in this study were specific for the relevant initiator caspases, as reported. To avoid activation of substrates by executioner caspases, all experiments were strictly limited to the first 30 min after induction of apoptosis by menadione. After loading, cells were washed and resuspended in calcium-free buffer solution. The isolated cells were placed on a Leica SP2 confocal microscope stage, and fluorescence was imaged over time (excitation 488 nm, emission 505–543 nm for caspase-2, -8, -9, or general caspase substrates; excitation 405 nm, emission 475–600 nm for caspase-10 and -12 substrates). Cells were then treated with 30 μM menadione. To examine colocalization of caspase activation and the position of mitochondria, cells were also loaded with MitoTracker Deep Red 633 (50 nM, excitation 633 nm, emission <650 nm) or tetramethyl rhodamine methyl ester (100 nM, excitation 543 nm, emission >600 nm) at 37°C for 15 or 20 min, respectively. Cells were then washed and resuspended in standard buffer solution. |
| References | |
| Additional Infomation |
Although the severity of experimental pancreatitis is correlated with the ratio of apoptosis to necrosis, the cell death program executed in the pancreas under pathological conditions remains incompletely elucidated. We elucidated the mechanism by which the oxidative stressor menadione induces apoptosis in pancreatic acinar cells. Real-time monitoring of initiating caspase activity showed that within 30 minutes of menadione administration, 66% of cells activated caspase-9, and 15% activated caspase-8, but no activation of caspase-2, -10, or -12 was detected. Interestingly, inhibition of caspase-9 activation increased caspase-8 activation. The half-maximal activation (t0.5) of caspase-9 occurred within approximately 2 minutes and was localized in or near the mitochondria; while the t0.5 of caspase-8 occurred approximately 26 minutes after menadione treatment and was uniformly distributed throughout the cell. The calcium chelator BAPTA or the mitochondrial permeability transition pore inhibitor ponkelidine completely blocked the activation of caspase-9, but had no effect on the activation of caspase-8. In contrast, lysosomal disruption (pre-incubation with cathepsin C substrate Gly-Phe β-naphthamide), loss of lysosomal acidity (bafloxacin A1), or inhibition of cathepsin L or D blocked the activation of caspase-8, but had no effect on the activation of caspase-9. We used the pepsin inhibitor A-BODIPY FL conjugate to demonstrate that cathepsin D translocates from lysosomes in the presence of menadione. We concluded that the oxidative stressor menadione induces two independent apoptotic pathways in pancreatic acinar cells: one is the classic mitochondrial calcium-dependent pathway, which is rapidly initiated in most cells; the other is a slower, caspase-8-mediated pathway that depends on the lysosomal activity of cathepsins and is initiated when the caspase-9 pathway is inhibited. [1]
|
| Molecular Formula |
C74H86N10O25
|
|---|---|
| Molecular Weight |
1515.53
|
| Exact Mass |
1514.576558
|
| PubChem CID |
171713852
|
| Sequence |
(Z-Ile-Glu-Thr-Asp)2-Rhodamine 110
|
| SequenceShortening |
(Z-IETD)2-Rhodamine 110
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| Appearance |
Typically exists as solids at room temperature
|
| Hydrogen Bond Donor Count |
16
|
| Hydrogen Bond Acceptor Count |
24
|
| Rotatable Bond Count |
41
|
| Heavy Atom Count |
109
|
| Complexity |
2960
|
| Defined Atom Stereocenter Count |
12
|
| SMILES |
CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(=O)O)C(=O)NC1=CC2=C(C=C1)C(=C3C=CC(=CC3=[O+]2)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)OCC4=CC=CC=C4)C5=CC=CC=C5C(=O)[O-])NC(=O)OCC6=CC=CC=C6
|
| InChi Key |
DSMSOMVESVLVDG-TVEQXDRTSA-N
|
| InChi Code |
InChI=1S/C74H86N10O25/c1-7-37(3)60(83-73(105)107-35-41-17-11-9-12-18-41)68(99)77-49(27-29-55(87)88)64(95)81-62(39(5)85)70(101)79-51(33-57(91)92)66(97)75-43-23-25-47-53(31-43)109-54-32-44(24-26-48(54)59(47)45-21-15-16-22-46(45)72(103)104)76-67(98)52(34-58(93)94)80-71(102)63(40(6)86)82-65(96)50(28-30-56(89)90)78-69(100)61(38(4)8-2)84-74(106)108-36-42-19-13-10-14-20-42/h9-26,31-32,37-40,49-52,60-63,85-86H,7-8,27-30,33-36H2,1-6H3,(H14-,75,76,77,78,79,80,81,82,83,84,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106)/t37-,38-,39+,40+,49-,50-,51-,52-,60-,61-,62-,63-/m0/s1
|
| Chemical Name |
2-[3,6-bis[[(2S)-3-carboxy-2-[[(2S,3R)-2-[[(2S)-4-carboxy-2-[[(2S,3S)-3-methyl-2-(phenylmethoxycarbonylamino)pentanoyl]amino]butanoyl]amino]-3-hydroxybutanoyl]amino]propanoyl]amino]xanthen-10-ium-9-yl]benzoate
|
| Synonyms |
Z-IETD-R110; Z-IETD R110;
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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)
|
| Solubility (In Vitro) |
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
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|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.6598 mL | 3.2992 mL | 6.5984 mL | |
| 5 mM | 0.1320 mL | 0.6598 mL | 1.3197 mL | |
| 10 mM | 0.0660 mL | 0.3299 mL | 0.6598 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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