Macrolide
Bacterial 50S ribosomal subunit (binding to 23S rRNA nucleotides G748 and A2058) [1]
Bacterial ribosome (common binding site for veterinary macrolides, involved in protein synthesis inhibition) [3]

Tylosin binds to the bacterial ribosomal 50S subunit's 23S rRNA to produce antibacterial effects [1].
With minimum inhibitory concentrations (MICs) of 64 μg/mL, 32 μg/mL, 512 μg/mL, and 1 μg/mL for M. haemolytica 11935, P. multocida 4407, E. coli ATCC 25922 and E. coli AS19rlmAI, respectively, tylosin also inhibits the growth of Gram-negative strains[3].

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Tylosin resistance is conferred by the synergistic action of single methylations at 23S rRNA nucleotides G748 and A2058; neither methylation alone is sufficient for resistance. This synergistic resistance mechanism is specific to tylosin and mycinamycin (possessing sugars extending from the 5- and 14-positions of the macrolactone ring) and not observed for macrolides like carbomycin, spiramycin, or erythromycin [1]
- At a concentration of 0.2 g/L, tylosin reduces total volatile fatty acids and lactobacilli, increases Clostridium cluster I after 6 hours of incubation with canine fecal microbiota; throughout the 24-hour incubation, it elevates pH values, spermidine, and E. coli levels. When combined with prebiotics (FOS, GOS, XOS) at 1 g/L, the prebiotics counteract some undesirable effects of tylosin, such as the decrease in lactobacilli and Clostridium cluster XIVa [2]
- As a 16-membered macrolide veterinary antibiotic, tylosin inhibits bacterial protein synthesis by binding to the ribosome, sharing a common ribosomal binding site with other veterinary macrolides (tildipirosin, tilmicosin, tulathromycin) [3]

Animals treated with lipopolysaccharide (LPS) showed an increase in IL-10 and a general suppression of elevated TNF-α and IL-1β levels when given tylosin (10–500 mg/kg; s.c.)[4].

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In healthy mice, tylosin at doses of 10 mg/kg and 100 mg/kg does not induce cytokine production; at 500 mg/kg, it has no effect on TNF-α or IL-1β production but induces IL-10 production [4]
- In lipopolysaccharide (LPS)-treated mice, all tested doses of tylosin (10 mg/kg, 100 mg/kg, 500 mg/kg) reduce elevated serum TNF-α and IL-1β levels and increase IL-10 levels, exhibiting an immunomodulatory effect [4]

Methyltransferase activity and resistance validation assay: Construct bacterial strains expressing TlrB (methyltransferase targeting G748) and/or TlrD (methyltransferase targeting A2058) methyltransferases. Incubate the strains with tylosin at relevant concentrations. Determine the minimum inhibitory concentration (MIC) of tylosin against the strains to assess whether single or combined methylations at G748 and A2058 confer resistance. Verify the specificity of the resistance mechanism by testing other macrolides (carbomycin, spiramycin, erythromycin, mycinamycin) [1]
- Ribosomal protein synthesis inhibition assay: Isolate bacterial ribosomes (e.g., from Escherichia coli or veterinary pathogenic bacteria like Mannheimia haemolytica, Pasteurella multocida). Prepare a cell-free protein synthesis system containing the ribosomes, mRNA, aminoacyl-tRNAs, and other necessary factors. Add tylosin to the system and incubate under optimal conditions. Monitor the synthesis of target proteins (e.g., via radioactive labeling or fluorescent detection) to evaluate the inhibitory effect of tylosin on ribosomal protein synthesis [3]

Canine fecal microbiota in vitro incubation assay: Collect feces from healthy adult dogs and prepare a fecal suspension. Mix the suspension with the residue of in vitro digested dry dog food in flasks. Set up eight treatment groups: control (no additives), tylosin (0.2 g/L), FOS (1 g/L), GOS (1 g/L), XOS (1 g/L), tylosin + FOS, tylosin + GOS, tylosin + XOS (five flasks per group). Incubate the flasks in an anaerobic chamber at 39 °C. Collect samples at 6 and 24 hours to analyze microbial composition (log₁₀ copies DNA/ng DNA) and metabolic parameters (pH, volatile fatty acids, biogenic amines) [2]
- Bacterial protein synthesis inhibition cell assay: Culture bacterial cells (e.g., Mannheimia haemolytica, Pasteurella multocida) in appropriate growth media. Add tylosin at gradient concentrations and incubate for a specified period. Measure the bacterial protein content or detect the expression of specific proteins to assess the inhibitory effect of tylosin on bacterial protein synthesis [3]

Animal Model: Balb/C mice (2-3 months old, 20-25 g)[4]
Dosage: 10 mg/kg, 100 mg/kg, 500 mg/kg
Administration: Subcutaneous injection
Result: raised the levels of IL-10 in mice treated with 250 µg of LPS, but decreased the elevated levels of TNF-α and IL-1β.

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Mouse cytokine response assay: Divide mice into seven groups: positive control group (injected with LPS), and six treatment groups (10 mg/kg tylosin, 100 mg/kg tylosin, 500 mg/kg tylosin, 10 mg/kg tylosin + LPS, 100 mg/kg tylosin + LPS, 500 mg/kg tylosin + LPS). Administer tylosin and LPS concurrently via appropriate routes. Collect serum samples at 0, 1, 2, 3, 6, 12, and 24 hours after treatment. Determine serum TNF-α, IL-1β, and IL-10 levels using enzyme-linked immunosorbent assay (ELISA) [4]

Absorption, Distribution and Excretion
This study employed a single-dose, randomized, parallel design to investigate the pharmacokinetics and oral bioavailability of tylosin tartrate and tylosin phosphate in broilers. Both tylosin formulations were administered orally and intravenously at a dose of 10 mg/kg body weight. Broilers were fasted overnight before administration (n=10 per group). A series of blood samples were collected at different time points within 24 hours after administration. The concentration of tylosin in chicken plasma was determined using high-performance liquid chromatography (HPLC). The concentration-time curve of tylosin in plasma for each chicken was analyzed using 3P97 software. After oral administration, the pharmacokinetics of tylosin best conformed to a one-compartment open model for the first absorption process. After intravenous administration, the pharmacokinetics of tylosin best conformed to a two-compartment open model, with no significant difference between tylosin tartrate and tylosin phosphate. Following oral administration, significant differences were observed in the Cmax (0.18±0.01 and 0.44±0.09, respectively) and AUC (0.82±0.05 and 1.57±0.25, respectively) of tylosin phosphate and tylosin tartrate. The calculated oral bioavailability (F) of tylosin tartrate and tylosin phosphate were 25.78% and 13.73%, respectively. In conclusion, we can reasonably conclude that tylosin tartrate is absorbed better than tylosin phosphate after oral administration.
/Milk/ This study aimed to determine the pharmacokinetics of tylosin and tilmicosin in the serum and milk of healthy Holstein cows (n = 12) and to reassess the residues in milk. Following intramuscular injection of tylosin, the maximum concentrations (Cmax) in serum and breast milk were 1.30 ± 0.24 μg/mL and 4.55 ± 0.23 μg/mL, respectively; the times to peak concentration (tmax) were at 2 hours and 4 hours, respectively; and the elimination half-lives were 20.46 ± 2.08 hours and 26.36 ± 5.55 hours, respectively. Following subcutaneous injection of tilmicosin, the Cmax in serum and breast milk were 0.86 ± 0.20 and 20.16 ± 1.13 μg/mL, respectively; the tmax were at 1 hour and 8 hours, respectively; and the elimination half-lives were 29.94 ± 6.65 hours and 43.02 ± 5.18 hours, respectively. The AUC_milk/AUC_serum and Cmax_milk/Cmax_serum ratios used to assess the rate of drug entry into milk were 5.01±0.72 and 3.61±0.69 for tylosin, and 23.91±6.38 and 20.16±1.13 for tilmicosin, respectively. In summary, it can be concluded that the concentration of tylosin in milk after parenteral administration is higher than expected, similar to tilmicosin, and requires a longer withdrawal period than reported. This study determined the bioavailability and pharmacokinetic characteristics of tylosin after oral and intravenous administration of 10 mg/kg to broilers. The bioavailability (F%) calculated by comparing the AUC values of oral and intravenous administration was 30%–34%. Following intravenous injection, tylosin rapidly distributes in the body, with an elimination half-life of 0.52 hours, a volume of distribution (Vd) of 0.69 L/kg, and a clearance (Cl) of 5.30 ± 0.59 mL/min/kg. After oral administration, the volume of distribution (Vd = 0.85 L/kg) is similar to that after intravenous injection, but the elimination half-life is 2.07 hours, four times that of the intravenous clearance (Cl = 4.40 ± 0.27 mL/min/kg). The time to peak concentration (tmax) after oral administration of tylosin is 1.5 hours, indicating that supplementation with this antibiotic via drinking water is the preferred method in broilers. However, the peak plasma concentration (Cmax = 1.2 μg/ml) after oral administration of tylosin is relatively low, suggesting that the dosage of this antibiotic in broilers should be higher than in other food-producing animals.
/Milk/ Antibiotic residues in milk exceeding tolerable levels can interfere with dairy processing and pose potential health risks to consumers. Residue avoidance programs include adherence to withdrawal periods indicated on labels. Residues in milk after antibiotic treatment are influenced by drug type, dosage, route of administration, body weight, and mammary gland health. Compositional changes occurring during intramammary infection (IMI) can affect antibiotic excretion in milk, thus altering withdrawal periods. This study aimed to validate the sensitivity and specificity of a qualitative microbiological method (Charm AIM-96) for detecting tylosin in bovine mixed milk and to determine the impact of subclinical intramammary infection on tylosin excretion after intramuscular injection. To validate this method, we used two groups of approximately 120 dairy cows each; one group received a single intramuscular injection of 20 mg/kg tylosin tartrate, and the other served as an untreated control group. The sensitivity and specificity of this method were 100% and 94.1%, respectively. To determine the effect of subclinical intramuscular infection on tylosin excretion, seven dairy cows in each of two groups (one group with a somatic cell count ≤250,000 cells/ml and the other with a SCC ≥900,000 cells/ml) received a single intramuscular injection of 20 mg/kg tylosin tartrate. Milk samples were collected every 12 hours for 10 consecutive days post-treatment. The mean time to tylosin excretion in milk was 5 days in the low somatic cell count group and 9 days in the high somatic cell count group (P < 0.0001). Changes in the composition of high somatic cell count (SCC) cows likely affect the pharmacokinetic characteristics of tylosin, prolonging the antibiotic's residence time in milk and thus influencing the withdrawal period. For more complete data on the absorption, distribution, and excretion of tylosin (10 parameters), please visit the HSDB records page.

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Metabolism/Metabolites
The tylosin biosynthesis (tyl) gene cluster of Streptomyces fradiae contains several accessory genes that encode functions typically related to primary metabolism. The deletion of these genes does not cause the strain to lose viability because equivalent genes are present elsewhere in the genome (presumably for maintaining "housekeeping" functions). The tyl gene cluster also contains two genes that encode products that are not identical to any protein in the database. Two accessory genes, metF (encoding N5,N10-methylenetetrahydrofolate reductase) and metK (encoding S-adenosylmethionine synthase), are located flanking an "unknown" gene (orf9) within the tyl gene cluster. In S. fradiae strains, the disruption of all three genes reduces tylosin production, but this effect is masked in media supplemented with glycine betaine (which provides methyl groups to the tetrahydrofolate pool). Clearly, one consequence of the recruitment of accessory genes to the tyl gene cluster is enhanced transmethylation capacity in secondary metabolic processes. Susceptibility studies of pathogenic Nocardia to the macrolide antibiotics chalcoxim and tylosin showed that most tested Nocardia species were highly resistant to both antibiotics, but Nocardia asteroides (N. nova) showed moderate susceptibility. Nocardia asteroides IFM 0339 converts these macrolide antibiotics into inactive metabolites via glycosylation at the 2'-OH site or glycosylation and reduction at the 20-formyl site. The structures of these metabolites were determined by NMR and mass spectrometry data to be 2'-[O-(β-D-glucopyranosyl)]chalcoxim (2), 2'-[O-(β-D-glucopyranosyl)]tylosin (5), and 20-dihydro-2'-[O-(β-D-glucopyranosyl)]tylosin (4). Tylosin is produced by Streptomyces fradiae through polyketide metabolism and the synthesis of three deoxyhexoses, with mekaminose being the first glycoside added to the polyketide aglycone tylosin (prototylosin). Previously, disruption of the gene encoding the linking of mycosamine glycosides to the glycoside ligand (tylMII) unexpectedly inhibited the accumulation of the glycoside ligand, suggesting a possible link between polyketide metabolism and deoxyhexose biosynthesis in Streptomyces fradiae. However, another explanation could not be ruled out at the time: the expression of other downstream genes not related to mycosamine glycoside metabolism might also contribute to this phenomenon. This paper shows that disruption of any one of the four genes specifically involved in mycosamine glycoside biosynthesis (tylMI-III and tylB) produces a similar response, confirming that the production of mycosamine glycoside-tylosin directly affects polyketide metabolism in Streptomyces fradiae. Under similar conditions, when mycosamine glycoside biosynthesis is specifically blocked by gene disruption, exogenous addition of glycosylated tylosin precursors can restore tylosin accumulation. Furthermore, certain other macrolide antibiotics with non-tylosin biosynthetic pathways have also been found to have similar qualitative effects. Comparing the structures of these stimulating macrolide antibiotics will help in studying their stimulatory mechanisms. In Streptomyces fradiae, tylosin biosynthesis involves three glycosyltransferases. The first sugar added to the polyketide aglycone (tylosin lactone) is macamiose, and the gene encoding the macamiose glycosyltransferase is orf2 (tylM2). However, under conditions that are generally favorable for tylosin production, targeted disruption of orf2 does not lead to the accumulation of tylosin lactone; instead, tylosin lactone synthesis is almost completely inhibited. This may be partly due to the polar effect of downstream gene expression of orf2, particularly orf4 (ccr), which encodes crotonyl-CoA reductase, an enzyme that provides the 4-carbon extension unit for polyketide metabolism. However, this is not the whole story, as tylosin production recovered to approximately 10% of wild-type levels when orf2 was reintroduced into the deletion strain. When glycosylated precursors of tylosin were added to the deletion strain, they were converted to tylosin, confirming that two of the three glycosyltransferase activities associated with tylosin biosynthesis remained intact. Interestingly, however, tylosin lactone also accumulated under these conditions, and the accumulation was significantly less when tylosin was added in similar fermentations. The conclusion is that glycosylated macrolide antibiotics significantly promote the metabolism of polyketides in Streptococcus fradiae.
More metabolite/metabolite (complete) data for tylosin (6 metabolites in total) can be found on the HSDB record page.

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Biological half-life
Bioavailability and pharmacokinetic properties of tylosin were determined in broilers after oral and intravenous administration at a dose of 10 mg/kg. ...After intravenous injection, the elimination half-life of tylosin is 0.52 hours. After oral administration, the elimination half-life of tylosin is 2.07 hours. It has been reported that the elimination half-life of tylosin is 54 minutes in small animals, 139 minutes in newborn calves, and 64 minutes in calves aged 2 months and older.

[1]. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy. Proc Natl Acad Sci U S A. 2002 Nov 12; 99(23): 14658-14663.

[2]. In Vitro Evaluation of the Effects of Tylosin on the Composition and Metabolism of Canine Fecal Microbiota. Animals (Basel). 2020 Jan; 10(1): 98.

[3]. Inhibition of Protein Synthesis on the Ribosome by Tildipirosin Compared with Other Veterinary Macrolides. Antimicrob Agents Chemother. 2012 Nov; 56(11): 6033-6036.

[4]. Effects of tylosin on serum cytokine levels in healthy and lipopolysaccharide-treated mice. Acta Vet Hung. 2010 Mar;58(1):75-81.

Tylosin is a macrolide antibiotic with the structure tylosin, where two hydroxyl groups are attached to a monosaccharide and a disaccharide moiety, respectively. It is naturally found in the fermentation products of Streptomyces fradiae. Tylosin can be used as a bacterial metabolite, allergen, exogenous substance, and environmental pollutant. It is an aldehyde, disaccharide derivative, enone, leucinogen, monosaccharide derivative, and macrolide antibiotic. Its structure is similar to tylosin. It is the conjugate base of tylosin(1+). Tylosin is a bacteriostatic macrolide antibiotic and feed additive used in veterinary medicine. It has broad-spectrum antibacterial activity against Gram-positive bacteria and is also effective against some Gram-negative bacteria. Tylosin is a fermentation product of Streptomyces fradiae.
Tylosin has been reported to exist in bovine (Bos taurus), Streptomyces fradiae, and Streptomyces venezuelae, and relevant data are available.
Tylosin is a macrolide antibiotic obtained from Streptomyces fradiae cultures. This drug is effective against a variety of microorganisms in animals but ineffective against humans.
See also: tylosin tartrate (salt form); tylosin phosphate (salt form); monensin; tylosin (component)...see more...

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Mechanism of Action
Tylosin is a 16-membered ring macrolide antibiotic. This study investigated its inhibitory effect on peptide bond formation in a model system derived from Escherichia coli. In this cell-free system, a peptide bond is formed between puromycin (receptor substrate) and AcPhe-tRNA (donor substrate) bound to the P site of the poly(U)programmed ribosome. Studies have shown that tylosin, as a slow-binding, slow-reversible inhibitor, can inhibit the reaction of puromycin. Detailed kinetic analysis revealed that tylosin (I) reacts rapidly with complex C (i.e., the AcPhe-tRNA·poly(U)·70S ribosome complex) to form encounter complex CI, followed by slow isomerization of CI into a tight complex CI inactive to puromycin. These processes can be described by the following reaction equation: C + I <==> (K(i)) CI <==> (k(4), k(5)) CI. The values of K(i), k(4), and k(5) are 3 μM, 1.5 min⁻¹, and 2.5 × 10⁻³ min⁻¹, respectively. The extremely low value of k(5) indicates that the inactivation of complex C by tylosin is almost irreversible. The irreversibility of tylosin's peptide bond formation is crucial for explaining the therapeutic properties of this antibiotic. Furthermore, the tylosin reaction is an effective tool for studying other macrolide antibiotics that, while not inhibiting the puromycin reaction, compete with tylosin for common ribosome binding sites. Therefore, this study combined the tylosin reaction with the puromycin reaction to investigate the mechanism of action of erythromycin. The results showed that erythromycin (Er), similar to tylosin, interacts with the complex C according to the kinetic scheme C + Er <==> (K(er)) CEr <==> (k(6), k(7)) CEr, forming a tight complex CEr that remains active against puromycin. The determination of K(er), k(6), and k(7) allowed us to classify erythromycin as a slow-binding ribosome ligand. Tylosin is a widely used antibiotic in veterinary medicine with potent antibacterial activity against Gram-positive bacteria [1]. The producing strain Streptomyces freundii protects itself from tylosin by differentially expressing four resistance determinants: tlrA, tlrB, tlrC, and tlrD. TlrB and TlrD encode methyltransferases that target the G748 and A2058 sites of 23S rRNA, respectively [1]. Tylosin has a 16-membered macrolide (tylosin lactone) ring and contains 5-macamiose and macamiose [3]. Tylosin has immunomodulatory effects at recommended therapeutic doses for infections [4].

C46H77NO17

916.1001

915.519

C, 60.31; H, 8.47; N, 1.53; O, 29.69

1401-69-0

Tylosin tartrate;74610-55-2;Tylosin phosphate;1405-53-4;Tylosin-d3

5280440

White to light yellow solid powder

1.2±0.1 g/cm3

980.7±65.0 °C at 760 mmHg

18-132ºC

546.9±34.3 °C

0.0±0.6 mmHg at 25°C

1.549

3.27

5

18

13

64

1560

21

O1[C@]([H])(C([H])([H])[H])C([H])([C@@]([H])(C([H])(C1([H])OC1([H])C([H])(C([H])([H])[H])[C@@]([H])(C([H])([H])C(=O)OC([H])(C([H])([H])C([H])([H])[H])C([H])(C([H])=C(C([H])([H])[H])C([H])=C([H])C(C([H])(C([H])([H])[H])C([H])([H])[C@]1([H])C([H])([H])C([H])=O)=O)C([H])([H])OC1([H])C([H])([C@@]([H])(C([H])(C([H])(C([H])([H])[H])O1)O[H])OC([H])([H])[H])OC([H])([H])[H])O[H])O[H])N(C([H])([H])[H])C([H])([H])[H])OC1([H])C([H])([H])C(C([H])([H])[H])([C@]([H])([C@]([H])(C([H])([H])[H])O1)O[H])O[H] |c:45,52|

WBPYTXDJUQJLPQ-VMXQISHHSA-N

InChI=1S/C46H77NO17/c1-13-33-30(22-58-45-42(57-12)41(56-11)37(52)26(5)60-45)18-23(2)14-15-31(49)24(3)19-29(16-17-48)39(25(4)32(50)20-34(51)62-33)64-44-38(53)36(47(9)10)40(27(6)61-44)63-35-21-46(8,55)43(54)28(7)59-35/h14-15,17-18,24-30,32-33,35-45,50,52-55H,13,16,19-22H2,1-12H3/b15-14+,23-18+/t24-,25+,26-,27-,28+,29+,30-,32-,33-,35+,36-,37-,38-,39-,40-,41-,42-,43+,44+,45-,46-/m1/s1

2-((4R,5S,6S,7R,9R,11E,13E,15R,16R)-6-(((2R,3R,4R,5S,6R)-5-(((2S,4R,5S,6S)-4,5-dihydroxy-4,6-dimethyltetrahydro-2H-pyran-2-yl)oxy)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-16-ethyl-4-hydroxy-15-((((2R,3R,4R,5R,6R)-5-hydroxy-3,4-dimethoxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)methyl)-5,9,13-trimethyl-2,10-dioxooxacyclohexadeca-11,13-dien-7-yl)acetaldehyde

Fradizine; Tylocine; Tylosin; Tylosin A; Tilosina; Tylan; Vubityl 200.

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)

DMSO : ≥ 100 mg/mL (~109.16 mM)
H2O : ~0.67 mg/mL (~0.73 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.73 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.73 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)