Integrin receptor

The whole surface modification process using Arg-Gly-Asp-Ser causes the αvβ3 integrin to be upregulated. Cellular antioxidant capabilities that are Arg-Gly-Asp-dependent are abolished when the phosphatidylinositol 3-pathway is blocked [1]. reactions from procaspase-3, -8, and -9. The activating action of Arg-Gly-Asp-Ser-peptide binding to survivin is located at the C-terminal of survivin and has a high affinity (Kd 27.5 μM). Survivin response and Arg-Gly-Asp-Ser appear to be important factors since survivin proliferation with certain siRNA causes Arg-Gly-Asp-Ser to lose its antimitotic function in cells [4].

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We tested the hypothesis that Arg-Gly-Asp-SerRGDS peptides regulate osteoblast survival in culture. Osteoblast-like MC3T3-E1 cells were allowed to attach to RGDS peptides that had been tethered to a silicone surface utilizing a previously described grafting technique. The RGDS-modified surface caused up-regulation of alpha(v)beta(3) integrin. We noted that there was an increase in expression of activated focal adhesion kinase and activated Akt. There was no change in the expression level of the anti-apoptotic protein Bcl-2, the pro-apoptotic protein Bad, or the inactivated form of Bad, pBad. Attachment to the RGDS-treated membrane completely abolished apoptosis induced by staurosporine, the Ca(2+).P(i) ion pair, and sodium nitroprusside. However, the surface modification did not interfere with apoptosis mediated by the free RGDS peptide or serum-free medium. When the activity of the phosphatidylinositol 3-kinase pathway was inhibited, RGDS-dependent resistance to apoptosis was eliminated. These results indicated that the binding of cells to RGDS abrogated apoptosis via the mitochondrial pathway and that the suppression of apoptosis was dependent on the activity of phosphatidylinositol 3-kinase.[1]

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In the present study we show for the first time that RGDS-peptide is internalized in melanoma cells in a time-dependent way and exerts strong anti-proliferative and pro-apoptotic effects independently from its extracellular anti-adhesive action. RGES control-peptide did not show biological effects, as expected; nevertheless it is internalized, although with slower kinetics. Survivin, a known cell-cycle and survival-regulator is highly expressed in melanoma cells. Co-immunoprecipitation assays in cell lysates and overlay assays with the purified proteins showed that Arg-Gly-Asp-Ser/RGDS interacts with survivin, as well as with procaspase-3, -8 and -9. RGDS-peptide binding to survivin was found to be specific, at high affinity (Kd 27.5 muM) and located at the survivin C-terminus. RGDS-survivin interaction appeared to play a key role, since RGDS lost its anti-mitogenic effect in survivin-deprived cells with a specific siRNA. Conclusions: Arg-Gly-Asp-Ser/RGDS inhibits melanoma growth with an adhesion-independent mechanism; it is internalized in melanoma cells and specifically interacts with survivin. The present data may indicate a novel role of RGDS-containing peptides physiologically released from the extracellular matrix and may suggest a possible novel anti-proliferation strategy in melanoma [4].

Four hours after LPS, Arg-Gly-Asp-Ser (2.5 or 5 mg/kg, 1 hour prior to LPS) dramatically reduced LPS-induced MMP-9 activity in BAL fluid. One hour prior to and four hours following LPS administration, the intraperitoneal injection of Arg-Gly-Asp-Ser (1, 2.5, or 5 mg/kg) suppresses the increase in TNF-α and MIP-2 levels in BAL fluid caused by LPS [2]. Tumor necrosis factor (TNF)-α and macrophage-stimulating protein (MIP)-2 production, as well as myeloperoxidase (MPO) and NF-κB activities, can all be markedly decreased by the Arg-Gly-Asp-Ser peptide [3].

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A pretreatment with Arg-Gly-Asp-Ser/RGDS inhibited LPS-induced increases in neutrophil and macrophage numbers, total protein levels and TNF-alpha and MIP-2 levels, and matrix metalloproteinase-9 activity in bronchoalveolar lavage (BAL) fluid at 4 or 24 h post-LPS treatment. RGDS inhibited LPS-induced phosphorylation of focal adhesion kinase and MAP kinases, including ERK, JNK, and p38 MAP kinase, in lung tissue. Importantly, the inhibition of the inflammatory responses and the kinase pathways were still evident when this peptide was administered 2 h after LPS treatment. Similarly, a blocking antibody against integrin alphav significantly inhibited LPS-induced inflammatory cell migration into the lung, protein accumulation and proinflammatory mediator production in BAL fluid, at 4 or 24 h post-LPS. Anti-beta3 also inhibited all LPS-induced inflammatory responses, except the accumulation of BAL protein at 24 h post-LPS. Conclusion: These results suggest that Arg-Gly-Asp-Ser/RGDS with high specificity for alphavintegrins attenuates inflammatory cascade during LPS-induced development of acute lung injury.[2]

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retreatment with synthetic Arg-Gly-Asp-Ser/RGDS peptide significantly improved LPS/D-GalN-induced mortality, and liver injury as determined by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, as well as pathological analysis. In addition, RGDS peptide significantly reduced tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2 production, and decreased myeloperoxidase (MPO) and NF-κB activity. Furthermore, Western blotting indicated that the levels of phospho-integrin β3, phospho-focal adhesion kinase (FAK) and phospho-p38 mitogen-activated protein kinases (MAPK) decreased with RGDS peptide pretreatment. Conclusion: Together, these data suggest that synthetic Arg-Gly-Asp-Ser/RGDS peptide protect against LPS/D-GalN-induced FHF by inhibiting inflammatory cells migration and blocking the integrin αVβ3-FAK-p38 MAPK and NF-κB signaling [3].

Preparation of RGD-treated Silicone Membranes [1]
RGD peptides were grafted to silicone surfaces utilizing a technique described previously based on a method originally reported by Dee et al. Briefly, 0.005-inch silicone sheets were exposed for 10 min to UV light/ozone to functionalize and oxidize the surface. The functionalized silicone surface was then modified by treatment with 0.2 mm 3-aminopropyltriethoxysilane in hexane for 45 min. The 3-aminopropyltriethoxysilane molecule reacted with the OH groups, generating an aminated surface. The silicone membranes were then sonicated in a hexane bath to remove excess reactants. The aminated membranes were incubated with 0.2 mm Arg-Gly-Asp-Ser/RGDS or RGES peptides in 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-ethyl morpholine. The carboxyl terminus of the peptide reacted with the amino group of the silane molecule, thereby forming a covalent link to the biomaterial surface. The membranes modified with the bound peptides were rinsed with N,N-dimethylformamide and distilled water. Non-bound peptides were removed by sonication in N,N-dimethylformamide for 15 min. Finally, the membranes with covalently attached peptides were sterilized in 75% ethanol. A control surface was engineered using RGES peptides using an identical procedure as described for Arg-Gly-Asp-Ser/RGDS. It has been shown that when Glu is substituted for Asp there is a profound loss of biological activity.

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Solid Phase Assay [4]
Bt-RGDS binding to recombinant GST-fusion proteins was evaluated by solid-phase assay (SPA) as previously described with modifications. Briefly, microtiter plates were coated with 175 nM (100 μl/well) recombinant GST-fusion proteins diluted in AC7.5 buffer for 4 h at RT. Wells were blocked in 30 mg/ml BSA (300 μL/well) overnight at 4°C, washed and incubated for 4 h at RT with increasing doses of bt-RGDS (from 1.75 to 1.75 mM), in the presence or in the absence of an excess of not-biotinylated RGDS/Arg-Gly-Asp-Ser, as specific competitor. After washing four times and incubation for 1 h at RT with 100 μl/well Vectastain-ABC Reagent, wells were stained with the ELISA Amplification System according to manufacturer's instructions and absorption at 495 nm (A495) was determined. Specific binding was computed by subtracting nonspecific from total binding at each concentration. Curve fit was carried out according to the following one-site specific bind equation.

Cell Culture [1]
Osteoblast-like MC3T3-E1 cells were used in this study. Our previous work has demonstrated that the apoptotic response of MC3T3-E1 cells is identical to that of primary human osteoblasts isolated from bone fragments. The cells were maintained in 10 ml of complete medium consisting of Dulbecco's minimum essential medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, and 50 μg/ml penicillin/streptomycin, pH 7.4. After the cells had reached confluence, they were released by treatment with 5 ml of 0.1% collagenase in Ca2+- and Mg+-free Hanks' buffered saline solution. The cells were then replated on the modified silicone membranes in 100-mm culture dishes and 12- or 24-well plates. Cultures were fed every other day with complete medium supplemented with 50 μg/ml ascorbic acid and 5 mm β-glycerophosphate. To evaluate cell death, osteoblast-like cells were plated on experimental (Arg-Gly-Asp-Ser/RGDS) and control (RGES) substrates in 12- or 24-well plates at a density of 50,000/well or 25,000/well, respectively. After 3 days in culture in complete media, the osteoblasts were incubated overnight with sublethal doses of staurosporine (0.1 and 0.5 μm), the Ca2+ (2.4 and 2.9 mm) and Pi (3 mm) ion pair, sodium nitroprusside (0.5 and 0.1 mm), or RGDS (1 mm and 5 mm) or were serum-starved. The ion pair, sodium nitroprusside, and staurosporine all induce cell death through the intrinsic pathway; RGDS and serum starvation probably kill osteoblasts by a mitochondria-independent pathway.

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Western Blot Analysis for Integrins [1]
After 0.5, 3, 24, and 72 h in culture on the prepared Arg-Gly-Asp-Ser/RGDS or RGES surfaces, cells were solubilized in 1% Nonidet P-40 lysis buffer. Protein concentration was determined using a BCA protein assay kit. 100 μg of protein was immunocomplexed with 1:50 dilution of anti-β3 integrin polyclonal IgG. After gentle mixing and incubation for 1 h on ice, 20 μg of protein A-Sepharose beads was added to the lysate. The mixture was centrifuged at 10,000 × g for 15 s at 4 °C; the pellet was washed five times with lysis buffer and resuspended in Laemmli sample buffer. The proteins were separated in 3–8% Tris acetate gel (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked in Tris-buffered saline with 5% skim milk powder. After washing in Tris-buffered saline, the blots were incubated with αv antibody (1:1000) overnight at room temperature. The blots were washed and incubated with horseradish peroxide-conjugated antibody for 1 h, and positive bands were detected using the ECL chemiluminescence kit and Kodak X-Omat blue film. GAPDH was used as a control to estimate protein loading on the gel. The concentration of the anti-GAPDH antibody was 2.4 μg/ml.

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Proliferation assay and apoptosis [4]
SK-MEL-110 were plated in 6-well plates (8 × 104 cells/well) on collagen-IV (50 μg/ml) and were grown for 24 h in complete medium in 5% CO2. Cells were then serum-starved for 24 h and subsequently treated with Arg-Gly-Asp-Ser/RGDS or RGES as control (500 μg/ml) dissolved in DMEM containing FGF-2 for 48 h. Then, cells were photographed, harvested by trypsin-EDTA and counted using hemacytometer. In other experiments, cells were pre-treated with a general caspase inhibitor (Z-VAD-FMK) (50 μM) for 2 h before RGDS treatment. To analyze cell cycle and sub-G1-phase, cells were fixed in ice-cold 70% ethanol and stained with propidium iodide (PI) at 10 μg/ml final concentration. Flow cytometry was performed on a Profile I flow cytometer.

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Biotinylated-RGDS (bt-RGDS) internalization and interaction with recombinant proteins [4]
To investigate peptides internalization, cells were treated with the biotinylated-RGDS (bt-RGDS) or biotinylated-RGES (bt-RGES) as control for different time-points, stained with phycoerythrin conjugated-avidin and analyzed by FACS. In additional experiments cells grown on collagen-IV were serum-starved for 48 h and treated for 24 h with biotin alone or with different doses of bt-RGDS (10-50-100 μg/ml). In other experiments cells were treated with 50 μg/ml bt-RGDS with an excess of 1 mg/ml un-labeled RGDS as specific competitor. Cells were washed to eliminate bt-RGDS bound to the membranes and a cytoplasmic extract was prepared as previously reported. Cytoplasmic lysates were spotted onto nitrocellulose, blocked with 5% milk in TPBS (0.1% Tween 20 in PBS) and incubated for 1 h at RT with a Vectastain ABC-peroxidase kit followed by chemiluminescence reaction and exposure to Kodak film. Interaction was quantified by densitometry and analyzed using the "Quantity one" software. [4]
In other experiments 40 μg of growing cells cytoplasmic extract, or increasing doses of recombinant human survivin (0.3-0.9-1.5-3-5 μg) or other recombinant proteins, as caspases-1 and -9, pro-caspase-9, fibronectin (0.9 μg) and BSA were spotted onto nitrocellulose, incubated for 4 h at RT with bt-RGDS (1 mg/ml) in the presence or absence of RGDS-excess (10 mg/ml) to measure the specific binding.

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Confocal microscopy [4]
SK-MEL-110 seeded on coverslips coated with collagen-IV and treated for 24 h with bt-RGDS (500 μg/ml), were washed as previously reported to eliminate the peptide bound to the membranes. Cells were fixed with 3% paraformaldehyde in PBS, pH 7.4, for 10 minutes, permeabilized with 0.1% Triton X-100 in PBS, pH 7.4, for 5 minutes at RT, and blocked for 30 minutes with BSA 2% in PBS, pH 7.4, at RT, followed by incubation with fluorescein avidin in PBS, pH 7.4, for 1 h at RT. After washing in 0.3% Triton X-100 in PBS, cells were incubated with PI at a final concentration of 5 μg/ml to visualize nuclei and analyzed using a Zeiss LSM 510 meta-confocal microscope. Laser power, beam splitters, filter settings, pinhole diameters and scan-mode were the same for all examined samples. To visualize active caspase-3 form, cells were treated with Arg-Gly-Asp-Ser/RGDS or RGES, fixed and incubated with a rabbit-monoclonal anti-active caspase-3 (1:150).

Mouse pharyngeal aspiration was performed as described by Rao et al. Animals were anesthetized with a mixture of ketamine and xylazine (45 mg/kg and 8 mg/kg, i.p., respectively). Test solution (30 μl) containing LPS (1.5 mg/kg) was placed posterior in the throat and aspirated into the lungs. Control mice were administrated sterile saline (0.9% NaCl). Animals were administered with Arg-Gly-Asp-Ser/RGDS or RGES peptide (1, 2.5 or 5 mg/kg, i.p.) once one hour before LPS treatment and sacrificed 4 h post-LPS Animals were also administered Arg-Gly-Asp-Ser/RGDS or RGES peptide (5 mg/kg, i.p.) once at different time points (1 h before or 2 h after LPS treatment) and sacrificed 24 h post-LPS. In addition, animals were administered with αvβ3-blocking mAbs, anti-αv, or anti-β3 (5 mg/kg, i.p.) once 1 h before and sacrificed 4 h post-LPS. Animals administered with these mAbs 2 h after LPS treatment were sacrificed 24 h post-LPS [2].

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To investigate the protection effect of Arg-Gly-Asp-Ser/RGDS peptides, mice were divided into four groups. Group I mice were intraperitoneally (i.p.) injected with normal saline as control. Group II mice were i.p. injected with Arg-Gly-Asp-Ser/RGDS peptide (5 mg/kg, dissolved in normal saline) alone. Group III mice were i.p. injected with LPS (50 μg/kg, dissolved in normal saline)/D-GalN (800 mg/kg, dissolved in saline). Group IV mice were pretreated with Arg-Gly-Asp-Ser/RGDS peptide (5 mg/kg) i.p. 30 min before an injection of LPS (50 μg/kg) and D-GalN (800 mg/kg). In some experiments, recombinant murine TNF-α (5 μg/kg) together with RGDS peptide (5 mg/kg) i.p. 30 min before an injection of LPS (50 μg/kg) and D-GalN (800 mg/kg).[3]
Six hours after LPS/D-GalN challenging, blood samples were collected, centrifuged at 3000 r.p.m. for 10 min to collect clear serum, for ALT, AST, and ELISA detection. And then, mice were sacrificed by decapitation. A portion of liver tissue was kept in 4% paraformaldehyde for histologic analysis. The rest of tissues were washed in cold saline and preserved at −80°C for further analysis of MPO, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and NF-κB activity. The survival rates were observed every 6 h within 48 h after LPS/D-GalN administration.[3]

[1]. Apoptosis and survival of osteoblast-like cells are regulated by surface attachment. J Biol Chem. 2005 Jan 21;280(3):1733-9.

[2]. Synthetic RGDS peptide attenuates lipopolysaccharide-induced pulmonary inflammation by inhibiting integrin signaled MAP kinase pathways. Respir Res. 2009 Mar 9;10:18.

[3]. Synthetic RGDS peptide attenuated lipopolysaccharide/D-galactosamine-induced fulminant hepatic failure in mice. J Gastroenterol Hepatol. 2014 Jun;29(6):1308-15.

[4]. Intracellular targets of RGDS peptide in melanoma cells. Mol Cancer. 2010 Apr 22;9:84.

Background: Synthetic peptides containing the RGD sequence inhibit integrin-related functions in different cell systems. Here, we investigated the effects of synthetic Arg-Gly-Asp-Ser (RGDS) peptide on key inflammatory responses to intratracheal (i.t.) lipopolysaccharide (LPS) treatment and on the integrin signaled mitogen-activated protein (MAP) kinase pathway during the development of acute lung injury. Methods: Saline or LPS (1.5 mg/kg) was administered i.t. with or without a single dose of RGDS (1, 2.5, or 5 mg/kg, i.p.), anti-alphav or anti-beta3 mAb (5 mg/kg, i.p.). Mice were sacrificed 4 or 24 h post-LPS.
The present study suggests that RGDS inhibits integrin (αvβ3)-dependent induction of inflammatory cell migration into the lungs, and proinflammatory mediator production. Our data also suggest that the protective effect of RGDS on pulmonary leakage is αv-dependent, but not β3. Importantly, posttreatment with RGDS was also highly effective at reducing these inflammatory responses correlated with its inhibitory effect on integrin signaled MAP kinase pathways. In addition, blockading the integrin-FAK-MAP kinase pathway with RGDS may reduce lung injury progression. Since RGDS has high specificity for αvintegrins and the in vivo efficacy to access target cells as a small-molecule, it may effectively attenuate inflammatory cascade during LPS-induced development of acute lung injury. [2]

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RGDS peptide has been reported to possess high efficiency against a series of malignancies such as recurrent glioblastoma, metastatic melanoma, and prostate cancer in clinical developments, it evokes our attentions to RGDS peptide, it evokes our attentions to RGDS peptide. In our study, we found that the synthetic RGDS peptide is a potential agent to protect against LPS/D-GalN-induced FHF. RGDS peptide improved LPS/D-GalN-induced survival rate, the extent of liver jury, inhibited macrophage and neutrophil migration, and TNF-α production by inhibiting the integrin αvβ3-FAK-p38 MAPK signaling and NF-κB signaling. The ability of RGDS to protect from LPS/D-GalN-induced FHF provides a potential therapeutic strategy to intervene in human liver injury diseases. In FHF, integrin signaling may contribute to disease pathogenesis and prognosis, which may become an important molecular target for treating inflammatory related hepatic disease.[3]

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Background: RGD-motif acts as a specific integrins-ligand and regulates a variety of cell-functions via extracellular action affecting cell-adhesion properties. However, increasing evidence identifies additional RGDS-functions at intracellular level. Previous reports show RGDS-internalization in endothelial cells, cardiomyocytes and lymphocytes, indicating intracellular targets such as caspase-8 and caspase-9, and suggest RGDS specific activity at cytoplasmic level. Given the role RGDS-peptides play in controlling proliferation and apoptosis in several cell types, investigating intracellular targets of RGDS in melanoma cells may un-reveal novel molecular targets and key pathways, potentially useful for a more effective approach to melanoma treatment.[4]

C17H28F3N7O10

547.44

547.1849

Arg-Gly-Asp-Ser;91037-65-9

139035042

H-Arg-Gly-Asp-Ser-OH.TFA; L-arginyl-glycyl-L-alpha-aspartyl-L-serine trifluoroacetic acid

RGDS

Typically exists as solid at room temperature

10

15

14

37

749

3

C(C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CO)C(=O)O)N)CN=C(N)N.C(=O)(C(F)(F)F)O

UMHUNRXMVGQLTE-YWUTZLAHSA-N

InChI=1S/C15H27N7O8.C2HF3O2/c16-7(2-1-3-19-15(17)18)12(27)20-5-10(24)21-8(4-11(25)26)13(28)22-9(6-23)14(29)30;3-2(4,5)1(6)7/h7-9,23H,1-6,16H2,(H,20,27)(H,21,24)(H,22,28)(H,25,26)(H,29,30)(H4,17,18,19);(H,6,7)/t7-,8-,9-;/m0./s1

(3S)-3-[[2-[[(2S)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-4-[[(1S)-1-carboxy-2-hydroxyethyl]amino]-4-oxobutanoic acid;2,2,2-trifluoroacetic acid

Arg-Gly-Asp-Ser (TFA); Arg-Gly-Asp-Ser TFA;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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.)