Potential Sources of Particulate Matter Impurities introduced during experimental operations: During weighing, aliquoting, or preparation, trace insoluble particles may be introduced due to environmental or consumable contamination (e.g., DMSO, EP tubes). If the stock solution is initially clear and the particles are minimal (visible under microscopy but not to the naked eye), the impact on experiments is typically negligible. Precipitation due to compound characteristics: CAY10566 is a hydrophobic small-molecule inhibitor. When the stock solution (50 mM in DMSO) is diluted into aqueous culture medium, precipitation may occur for the following reasons: Change in solvent polarity: Transition from highly polar DMSO to less polar aqueous medium reduces compound solubility. pH or ionic strength changes: Medium components (e.g., buffer salts, serum) may alter the local microenvironment, inducing crystallization. Temperature/CO₂ equilibration: Incubator conditions (37°C, 5% CO₂) may affect compound stability. Verification and Solutions Eliminating operational contamination: Ensure DMSO and consumables (EP tubes, pipette tips) are free of RNase/DNase and particulate contamination. Negative control: Prepare pure DMSO + medium alone to observe if particles appear. Strategies to improve solubility: Sonication: Briefly sonicate the working solution (5–10 sec, ice bath to prevent overheating) to check if particles dissolve. Adjust solvent ratio: Increase the final DMSO concentration to 0.2–0.5% (confirm cell tolerance). Test co-solvents (e.g., PEG-300, ethanol), but evaluate potential interference with the experimental system. Sterile filtration: Use a 0.22 μm filter to remove insoluble particles from the stock or working solution (negligible impact on concentration). Impact Assessment on Experiments If particles are trace impurities (not major compound precipitation), filtration will retain the actual CAY10566 concentration close to the theoretical value, with minimal effect on downstream experiments. If significant compound precipitation occurs, re-optimize solvent conditions (e.g., reduce working concentration, switch solvents). Key Considerations DMSO tolerance: Ensure the final concentration does not exceed 0.5% (routine cells tolerate ≤1%). Stability testing: Prepare solutions fresh before use to avoid long-term storage-induced precipitation.

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