PKA (EC50 = 360 nM)

Sp-8-Br-cAMPS sodium (0-100 nM) inhibits phagocytosis of X. nematophila and B. subtilis without affecting the bacterial numbers of each blood cell type[1]. Sp-8-Br-cAMPS sodium (0-1000 μM) dose-dependently inhibits Staphylococcal enterotoxin B (SEB)-induced T cell activation and the expression of cytokines IFN-γ, TNF-α, IL-2, and IL-4 via activation of PKA[2].

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Phagocytosis of X. nematophila was substantially impaired by the PKA activator in a concentration-dependent manner (Figure 3a), whereas inhibition of B. subtilis occurred only at high concentrations (Figure 3b). The granular cell levels with either bacterial species attached also diminished. Although plasmatocyte levels with X. nematophila declined, there was no change in levels with B. subtilis. The enzyme activator did not affect the number of bacteria per haemocyte type (X. nematophila: control, 1.7 ± 0.3 bacteria/plasmatocyte, 2.1 ± 0.3 bacteria/granular cell; 100 nmol/L Sp-8-Br-cAMPS, 1.3 ± 0.2 bacteria/plasmatocyte, 1.9 ± 0.3 bacteria/granular cell [P > 0.05]; B. subtilis: control, 2.0 ± 0.4 bacteria/plasmatocyte, 1.7 ± 0.3 bacteria/granular cell; 100 nmol/L Sp-8-Br-cAMPS, 1.4 ± 0.3 bacteria/plasmatocyte, 1.2 ± 0.2 bacteria/granular cell [P > 0.05]). However, a consistent pattern of decline in bacteria per haemocyte type was detected. Co-incubating haemocytes with 50 nmol/L PKA activator and increasing amounts of PKA inhibitor increased the levels of granular cells and plasmatocytes with both bacterial species (Figure 4). [1]

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On their own, the PKA modulators influenced haemocytic protein release. Increasing concentrations of the enzyme inhibitor increased protein release, whereas the increasing concentrations of the activator impaired protein release (Figure 5b). Rp-8-Br-cAMPS offset the inhibition of protein discharge caused by the PKA activator Sp-8-Br-cAMPS. [1]

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cAMP and PKA type I inhibits SEB-induced T cell activation and expression of IFN-γ [2]
To explore possible effects of cAMP and PKA type I on early functional events in immune activation, we measured the expression of the activation marker CD69 and production of IFN-γ after activation of PBMC with the superantigen SEB for 6 h. The expression of CD69 and IFN-γ were measured by flow cytometry in the presence and absence of the PKA agonist Sp-8-Br-cAMPS alone or in combination with the PKA type I selective antagonist Rp-8-Br-cAMPS. The cells were preincubated with cAMP analogs for 1 h to allow diffusion of the compounds, followed by activation with SEB. Brefeldin A was then added 1 h after activation with SEB, and the cultures were incubated for 5 h for cytokine accumulation. The expression of both CD69 and IFN-γ were markedly reduced by preincubation with 250 μM Sp-8-Br-cAMPS (agonist) in CD3+ T cells compared with control (Fig. 1, a and b). To clarify whether this inhibited cytokine production is mediated through PKA type I, associated with the TCR/CD3 and lipid rafts, or PKA type II, whose localization is mainly confined to the perinuclear and Golgi centrosomal regions in lymphocytes (17), we preincubated the cells with 250 μM Sp-8-Br-cAMPS in combination with 1000 μM of the PKA type I selective antagonist Rp-8-Br-cAMPS (Fig. 1,c). The antagonist reversed almost completely the inhibitory effect of the agonist, indicating that the inhibitory effect of cAMP in the early T cell activation is mainly due to activation of PKA type I. The antagonist had no effect on CD69 and IFN-γ expression alone (Fig. 1 d).

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The inhibitory effect of cAMP and PKA type I is not due to apoptosis [2]
To investigate whether the inhibitory effect of cAMP and PKA type I was due to induction of apoptosis, we evaluated the morphology of unstimulated and SEB-stimulated PBMC with and without preincubation with 1000 μM Sp-8-Br-cAMPS and with and without incubation with brefeldin A for the last 5 h of the incubation time by flow cytometry on forward and side scatter. The cultures were incubated for various periods of time ranging from 6 to 48 h and did not demonstrate the morphological changes characteristic of apoptosis, with decrease in forward scatter and increased side scatter, in the presence of the cAMP analog (data not shown). Furthermore, apoptosis was also assessed by Annexin VFITC labeling under the same experimental conditions (Fig. 2, culture incubated for 24 h is shown). The cAMP agonist did not induce any significant increase in annexin V binding as a marker of apoptotic cells in the resting (Fig. 2, a and b) or activated cultures (Fig. 2, c and d). In contrast, anisomycin used as control (Fig. 2, e and f) induced apoptosis and increased annexin V binding in both unstimulated and activated cultures. Similar data were obtained by propidium iodide exclusion (data not shown).

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cAMP and PKA type I inhibits SEB-induced expression of IFN-γ, TNF-α, IL-2, and IL-4 [2]
To examine whether the effects of cAMP and PKA type I were specific for IFN-γ expression, we measured the expression of various cytokines after SEB activation of PBMC with increasing concentrations of Sp-8-Br-cAMPS. We found that expression of all cytokines examined (IFN-γ, TNF-α, IL-2, and IL-4) was reduced in a concentration-dependent manner by the cAMP analog (Fig. 3 a), although the sensitivity to inhibition by the cAMP analog varied among the cytokines. In all cases, the cAMP-mediated inhibition was reversible with the PKA type I selective antagonist Rp-8-Br-cAMPS. We next measured the level of inhibition of cytokine expression with longer cytokine accumulation periods to see whether the effect was transient. Accumulation periods of 5, 15, and 25 h were conducted, and the level of inhibition was persistent and concentration dependent at all of these time points (data not shown).

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cAMP and PKA type I inhibits both CD4 and CD8 T cell activation independently of APC [2]
To further address the question of which T cell compartment is affected by cAMP in the inhibition of immune activation, we investigated the effects of cAMP and PKA type I on CD4 and CD8 T cells. As shown in Fig. 5, a and b, the expression of both IFN-γ and TNF-α was inhibited in a concentration-dependent manner. However, this experiment was performed with SEB stimulation of PBMC containing APC. To eliminate the possibility that the effects observed could be due to suppressive effects of cAMP on the function of APC, we isolated APC by adherence and loaded the cells with SEB followed by paraformaldehyde fixation. We then set up an autologous coculture with fixed, Ag-loaded APC, and an APC-depleted PBMC population at a 1:10 ratio. The coculture was incubated for 6 h before addition of brefeldin A. Cytokine accumulation was allowed to proceed for 14 h. The expression of IFN-γ was assessed by intracellular flow cytometry in CD4 and CD8 T cells and a concentration-dependent inhibition of cytokine expression was observed with increasing concentrations of Sp-8-Br-cAMPS showing that inhibition is independent of APC (Fig. 5 c).

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Coupled inhibition of cytokine expression and proliferation [2]
To further determine the extent of the cAMP-mediated inhibition of immune functions, we investigated the effects on proliferation of CD3, CD4, and CD8 T cells by the CFSE dilution assay. As Fig. 6 a shows, a concentration-dependent decrease in SEB-induced proliferation with increasing concentrations of Sp-8-Br-cAMPS is observed in all three cell populations. The inhibition was reversible with the PKA type I selective antagonist Rp-8-Br-cAMPS (data not shown). By combining the CFSE dilution assay with intracellular flow cytometry, we measured the effect of cAMP on both immune functions simultaneously. As shown in Fig. 6 b, there is a sequential reduction of CFSE fluorescence intensity for each cell division, with the highest fraction of IFN-γ-expressing T cells being in the later daughter cell generations. With increasing concentrations of Sp-8-Br-cAMPS, there was a profound reduction in IFN-γ expression and a marked suppression of proliferation shown by disappearance of the last two cell divisions with 1000 μM Sp-8-Br-cAMPS.

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cAMP and PKA type I inhibits Ag-specific immune responses [2]
We next examined the effect of cAMP and PKA type I on Ag-specific immune responses (Fig. 7 a). As seen with SEB-induced immune responses, we observed a concentration-dependent inhibition of CMV-induced IFN-γ expression by increasing concentrations of Sp-8-Br-cAMPS in both the CD4 and CD8 T cell populations. To induce optimal stimulation, the CD4 T cell population was stimulated by CMV-infected cell extracts, while the CD8 T cell population was stimulated by CMVpp65. The range of inhibition was from 60 to 80% in both cell populations. We also examined the effect of PKA type I activation on tuberculin PPD-induced T cell proliferation and IFN-γ production by combining the CFSE dilution assay with intracellular flow cytometry (Fig. 7 b). As observed with the SEB- and CMV-induced immune responses, the T cell proliferation as well as IFN-γ expression induced by tuberculin PPD declined in a concentration-dependent manner with Sp-8-Br-cAMPS. Furthermore, both T cell proliferation and IFN-γ expression were fully reversible with the cAMP antagonist Rp-8-Br-cAMPS (data not shown).

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cAMP activates Csk [2]
To assess the effect the cAMP agonist used in this study on the previously mapped mechanism for cAMP-PKA inhibition of T cell functions (11), we examined whether Sp-8-Br-cAMPS lead to an increase in Csk activity. Csk phosphotransferase activity was assessed in vitro after treatment with increasing concentrations with the cAMP analog resulting in a concentration-dependent increase in Csk activity with a >2-fold increase in the activity with the highest concentration used (Fig. 8).

Sp-8-Br-cAMPS sodium (50 nM) inhibits protein release from hemocytes and inhibits bacterial removal from the hemolymph, but does not affect the viability of G. mellonella larvae [1].

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Within 30 min of injecting Sp-8-Br-cAMPS alone, the total haemocyte counts increased compared with the PBS control larvae, whereas the PKA inhibitor lowered haemocyte levels (Figure 7). Nodules (1.2 ± 0.1 mm diameter) and extensive haemocyte aggregates (2 × 105/mL) were seen in the insects with inhibitor but not in the groups with the activator. Increasing amounts of the inhibitor decreased haemocyte counts in larvae containing a fixed amount of PKA activator. [1]

PKA biochemical assay [1]
To establish that the PKA modulators affected PKA activity and that PKA activity changed during haemocyte adhesion, a PKA biochemical assay was used. Chilled larvae were bled and 180 µL of haemolymph was added to 200 µL of anticoagulant23. Two hundred microlitres of haemocyte suspension was centrifuged (200 g, 2 min, 4°C), and the haemocyte pellet was washed twice in 100 µL PBS by centrifugation. The first set of pellets was resuspended in 20 µL PBS containing selected amounts of Rp-8-Br-cAMPS and/or Sp-8-Br-cAMPS. After 30 min incubation, the cells were washed by centrifugation three times in 1 mL PBS, lysed by vigorous pipetting, and assayed for PKA activity according to the manufacturer's instructions. The second haemocyte set was placed in 96-well microtitre plates containing glass coverslips (5 mm diameter). To prevent possible changes in PKA activity by haemocyte adhesion, the haemocytes were immediately lysed to serve as the nonattached haemocyte control group. After 30 min incubation at room temperature, the cells in other wells representing predominantly adhering haemocytes were lysed. The PKA activities of both groups were determined. A minimum of two samples for each of four replicates was used.

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Assessment of Csk activity [2]
Purified peripheral T cells (40 × 106 cells/ml in RPMI 1640) were incubated at 37°C for 30 min with or without the cAMP analog Sp-8-Br-cAMPS at indicated concentrations. Thereafter, cells were disrupted in lysis buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 50 mM n-β-octyl-glucoside, 10 mM NaPPi, 1 mM Na3VO4, 50 mM NaF, and 1 mM PMSF) and subjected to immunoprecipitation with anti-Csk Ab. After overnight incubation at 4°C, protein A-Sepharose was added and the incubation was continued for 1 h. Immune complexes were washed three times in lysis buffer and three times in Csk kinase assay buffer (50 mM HEPES and 5 mM MgCl2, pH 7.4), followed by Csk kinase assays and Western blot analysis. The tyrosine kinase activity of human Csk was measured as incorporation of [32P]phosphate into the synthetic polyamino acid poly(Glu,Tyr) 4:1. A standard protocol was followed with reaction volumes of 50 μl containing HEPES buffer (pH 7.4), 5 mM MgCl2, 200 μM [γ-32P]ATP (0.15 Ci/mmol), 200 μg/ml poly(Glu,Tyr), and immunoprecipitated Csk. The incubation temperature was 30°C, and the incubation time was 12 min.

Bacterial phagocytosis and haemocytes with associated bacteria [1]
Haemocyte monolayers were prepared by adding 60 µL of insect haemolymph collected from a total of six larvae that had been chilled on ice (15 min) to 1 mL of ice cold PBS (1.6 × 106 haemocytes/mL). Unless stated otherwise, the haemocytes were not washed free of plasma so that they more closely approximated the in vivo situation. Fifteen microlitres of the suspension was placed on endotoxin-free glass slides and was incubated at >95% relative humidity for 30 min to allow the haemocytes to adhere. Non-attached haemocytes were removed by rinsing the slides three times with 2 mL PBS. Fluorescent bacteria in PBS with and without selected concentrations of PKA modulators (PKA activator Sp-8-Br-cAMPS; PKA type I inhibitor RP-8-Br-cAMPS27) were added to the monolayers, producing a bacteria : haemocyte ratio of 10:1. The slides were incubated in high humidity with shaking (25 r.p.m.) for 1 h, after which time non-attached bacteria were removed by rinsing three times with PBS. Fluorescence of non-phagocytosed bacteria was quenched with trypan blue (0.2% w/v28). The haemocytes with bacteria were fixed in glutaraldehyde–formaldehyde vapour for 10 min and were mounted with glycerol (20% v/v) in PBS. Haemocytes, where required, were identified as granular cells or plasmatocytes29. The levels of total phagocytic haemocytes and plasmatocytes (expressed as a percentage of the total haemocytes and total plasmatocytes) were determined by fluorescence microscopy. The number of each haemocyte type with attached bacteria (attachment included phagocytosed bacteria and bacteria on the haemocyte surface) and number of attached bacteria per haemocyte type were determined using phase contrast microscopy. A minimum of 150 haemocytes of each type was examined for each of 10 replicates containing 10 samples.

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Haemocyte protein discharge [1]
Haemocytes free of haemolymph plasma were obtained by chilling 10 larvae on ice (15 min) and collecting 75 µL of the haemolymph in 1 mL of chilled anticoagulant. The haemocytes were washed three times by centrifugation to remove plasma and anticoagulant (200 g, 2 min). The pellet was then resuspended in cold PBS (5°C, 1 mL). Haemocyte suspensions (100 µL) were added to the wells of a 96-well microtitre plate containing circular (5 mm diameter) glass coverslips and PBS with and without selected concentrations of Rp-8-Br-cAMPS and Sp-8-Br-cAMPS or with a fixed level of activator and 10 and 50 nmol/L of inhibitor. Other wells contained buffer and PKA modulators with both bacterial species producing a 10:1 bacteria : haemocyte ratio. Bacteria with and without the enzyme modulators served as haemocyte-free controls. Plates were incubated at 22°C for 15–60 min, during which time samples were removed by pipetting and centrifuged (2000 g, 2 min). The total protein in the supernatant was determined. Ten replicates each containing two samples were used throughout the incubation.

PKA modulators in vivo [1]
Five replicates containing two groups of 10 larvae were injected at the base of the prothoracic leg with 10 µL PBS containing selected levels of Sp-8-Br-cAMPS and/or Rp-8-Br-cAMPS. The insects of one group were bled 30 min after injection, and the total haemocyte counts per individual were determined on a haemocytometer. Gross dissection was done on the remaining group 24 h after injection to detect nodules. The effects of the PKA modulators on the removal of B. subtilis and X. nematophila were similarly assessed with all test solutions containing 1 × 108 bacteria per 10 µL. Both bacterial and haemocyte levels were determined on a haemocytometer.

[1]. Protein kinase A affects Galleria mellonella (Insecta: Lepidoptera) larval haemocyte non-self responses. Immunol Cell Biol. 2005 Apr;83(2):150-9.

[2]. , Inhibition of antigen-specific T cell proliferation and cytokine production by protein kinase A type I. J Immunol. 2002 Jul 15;169(2):802-8.

We used the protein kinase A (PKA) specific activator Sp-8-Br-cAMPS and type I inhibitor Rp-8-Br-cAMPS alone and in combination to define the role of PKA in the non-self responses of larval Galleria mellonella haemocytes in vitro and in vivo. Active PKA depressed haemocyte responses whereas PKA inhibition enhanced activities, including bacterial phagocytosis, the number of haemocytes with adherent bacteria, bacterial-induced haemocytic protein release and haemocyte adhesion to slides in vitro, as well as in vivo bacterial removal from the haemolymph. Non-attached haemocytes had more PKA activity than attached haemocytes; therefore, active PKA limited haemocyte response to foreign materials. We found that (i) PKA inhibitor alone induced non-self responses, including haemocyte protein discharge and lowered haemocyte counts in vivo, and induced nodulation; (ii) the enzyme activator produced effects opposite to those of the inhibitor; and (iii) together, the modulators offset each others' effects and influenced haemocyte lysate PKA activity. These findings establish PKA as a mediator of haemocytic non-self responses. [1]

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cAMP inhibits biochemical events leading to T cell activation by triggering of an inhibitory protein kinase A (PKA)-C-terminal Src kinase pathway assembled in lipid rafts. In this study, we demonstrate that activation of PKA type I by Sp-8-bromo-cAMPS (a cAMP agonist) has profound inhibitory effects on Ag-specific immune responses in peripheral effector T cells. Activation of PKA type I inhibits both cytokine production and proliferative responses in both CD4(+) and CD8(+) T cells in a concentration-dependent manner. The observed effects of cAMP appeared to occur endogenously in T cells and were not dependent on APC. The inhibition of responses was not due to apoptosis of specific T cells and was reversible by a PKA type I-selective cAMP antagonist. This supports the notion of PKA type I as a key enzyme in the negative regulation of immune responses and a potential target for inhibiting autoreactive T cells. [2]

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We and others have shown that cAMP and PKA type I act as an acute inhibitor of TCR/CD3 signaling on the molecular level. In this study, we present data showing that cAMP through activation of PKA type I is a universal inhibitor of T cell function. Preincubation with Sp-8-Br-cAMPS inhibited Ag-induced expression of IFN-γ, TNF-α, IL-2, and IL-4 in a dose-dependent manner. The inhibition involved both CD4 and CD8 T cells, and the effect was independent of APC and did not induce apoptosis. Furthermore, the cAMP analog inhibited both SEB-induced proliferation and Ag-specific immune responses to CMV and tuberculin PPD. Coincubation with Rp-8-Br-cAMPS, which is a selective inhibitor of PKA type I, completely reversed the inhibitory effect of the PKA agonist Sp-8-Br-cAMPS. This indicates that the effects observed are due to activation of PKA type I and that inhibition of T cell function following cAMP incubation can be reversed in a timely manner with a cAMP antagonist.[2]

C10H10BRN5NAO5PS

446.15

444.922

1573115-90-8

24757345

Typically exists as solid at room temperature

2

10

1

24

542

4

C1[C@@H]2[C@H]([C@H]([C@@H](O2)N3C4=NC=NC(=C4N=C3Br)N)O)OP(=S)(O1)[O-].[Na+]

SKJLJCVVXRNYGJ-QKAIHBBZSA-M

InChI=1S/C10H11BrN5O5PS.Na/c11-10-15-4-7(12)13-2-14-8(4)16(10)9-5(17)6-3(20-9)1-19-22(18,23)21-6;/h2-3,5-6,9,17H,1H2,(H,18,23)(H2,12,13,14);/q;+1/p-1/t3-,5-,6-,9-,22?;/m1./s1

sodium;(4aR,6R,7R,7aS)-6-(6-amino-8-bromopurin-9-yl)-2-oxido-2-sulfanylidene-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-7-ol

925456-59-3; 1573115-90-8; Rp-8-bromo-Cyclic AMPS (sodium salt); Rp-8-bromo-Cyclic AMPS sodium salt; 8-Bromoadenosine 3',5'-cyclic monophosphothiaoate, Sp-isomer sodium salt; sodium;(4aR,6R,7R,7aS)-6-(6-amino-8-bromopurin-9-yl)-2-oxido-2-sulfanylidene-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-7-ol; 8-Bromoadenosine3',5'-CyclicMonophosphothioateSp-IsomerSodiumSalt; 8-bromo-adenosinecyclic3',5'-[hydrogen[P(R)]-phosphorothioate],monosodiumsalt;

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.)