Generally, an acidic stock solution is prepared (adjusted to pH ≈2 using HCl) with the aid of ultrasonic heating for dissolution. For storage, it is recommended to use the solution within 6 months when kept at -80°C, or within 1 month when stored at -20°C (protection from light and moisture is required). The working solution is then prepared by diluting the stock solution in PBS (pH adjusted to 2-3 with HCl), which should be used immediately and is not recommended for storage. However, since you require intraperitoneal injection, as a precautionary measure, we suggest first dissolving the compound in acidified water to prepare a high-concentration stock solution (e.g., 3 mg/mL). This can then be diluted 1000-fold with PBS or saline. After dilution, the solution will be essentially neutral and suitable for normal injection.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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Here’s a careful, step-by-step approach based on standard laboratory practices for preparing insulin for intraperitoneal (IP) injection in animal studies:

1. Choosing a Solvent

Sterile saline (0.9% NaCl) is commonly used and generally safe for IP injections.

PBS can be used, but avoid high phosphate concentrations or additives that might precipitate insulin.

Acidified water (e.g., 0.01 N HCl) is sometimes recommended for initial dissolution of lyophilized insulin because insulin is more soluble at slightly acidic pH. Once dissolved, the solution is often neutralized with PBS or saline to physiological pH (~7.2–7.4) before injection.

Practical approach:

1. Dissolve insulin first in a small volume of dilute acid (0.01 N HCl) to fully solubilize it.

2. Dilute with sterile saline to reach the desired working concentration (0.003 mg/mL).

3. Check that the final solution is clear; any precipitate indicates incomplete dissolution.

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2. Sterility Considerations

Work under sterile conditions (biosafety cabinet) and use sterile glassware and filters.

After preparation, filter the solution through a 0.22 µm sterile filter if possible.

3. Storage Conditions

Short-term (hours to a few days): Keep at 4 °C, tightly capped, protected from light.

Long-term: Aliquot into small volumes (e.g., 0.5–1 mL) and store at –20 °C or –80 °C. Avoid repeated freeze-thaw cycles, which degrade insulin.

Before injection, thaw gently on ice or at 4 °C and gently mix without vortexing.

4. Stability

Insulin in saline or PBS at 4 °C is usually stable for 1–2 weeks if protected from light and microbial contamination.

In frozen aliquots, insulin can remain stable for several months (often up to 6 months), though checking the manufacturer’s datasheet for HY-P0035 is recommended.

5. Additional Tips

Avoid shaking or vortexing vigorously; insulin is sensitive and can aggregate.

Prepare fresh working solutions whenever feasible to ensure activity.

Confirm final pH is near physiological before IP injection to prevent irritation.