RBM1-151 (20 μM, 1 h) was hydrolyzed by acidic ceramidinase to RBM1-151-NH2 in a cell-free system (using A375/AC cell lysis buffer) [1]. RBM1-151 (0–20 μM, 30 min) was hydrolyzed in acidic buffer with A375/AC cell lysis buffer (20 μg), and the kinetic parameters calculated by Michaelis-Menten analysis were appKm = 7.0 μM and appVmax = 99.3 nM/min [1]. RBM1-151 (5 μM, 3 h) showed higher hydrolysis (fluorescent product) in HEK293/NAAA cell lysis buffer (10 μg, overexpressing NAAA) than in HEK293/mock lysis buffer [1]. RBM1-151 (20 μM, 3 h) produced very little fluorescence in Farber disease (FD) cells (lacking AC), but significantly higher fluorescence in FD/AC cells (overexpressing AC) (in intact cells) [1]. RBM1-151 (20 μM, 3 h) showed higher hydrolysis in A375/AC cells (overexpressing AC) than in A375/WT cells [1]. Guidelines (The following are our recommended experimental protocols. These protocols are for guidance only and should be adjusted according to specific needs.) 1. Cell preparation 1.1 Suspension cells (e.g., AML cell lines: MM-6, HL-60): Culture cells in RPMI-1640 medium containing 20% FBS; adjust the cell density to 2 × 10⁴ cells per well before dispensing. 1.2 Adherent cells (e.g., melanoma cell lines: A375/AC, C8161; HEK293): Culture cells in Dulbecco modified Eagle medium (high glucose) containing 10% FBS and 1% penicillin/streptomycin; seed 2 × 10⁴ cells per well in a 96-well plate and incubate overnight to ensure cell adhesion. Note: For cell lines requiring antibiotic selection (e.g., using blastin and hygromycin for A375/AC), remove the antibiotics from the medium before the experiment to avoid interference. 2. RBM1-151 Incubation and Signal Detection 2.1 Add 50 μL of 20 μM RBM1-151 working solution (prepared in medium containing 20% FBS, final RBM1-151 concentration 20 μM) to each well. 2.2 Incubate at 37°C and 5% CO₂ for 1 hour. 2.3 Add 25 μL of 100% methanol to each well to terminate the reaction. 2.4 Immediately add 100 μL of NaIO4 solution (2.5 mg/mL, prepared in 100 mM glycine-NaOH buffer, pH 10.6) to each well. 2.5 Incubate at 37°C and 5% CO2 in the dark for 30 minutes. 2.6 Measure the fluorescence signal using a microtiter plate reader; subtract the background signal from wells containing only culture medium and RBM1-151 but no cells.

[1]. https://pubmed.ncbi.nlm.nih.gov/38369184/

[2]. https://pubmed.ncbi.nlm.nih.gov/39062935/

C28H41NO5

471.63

3077081-47-8

Typically exists as solids at room temperature

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.)