In my experiment, I needed to prepare a cholesterol (HY-N0322, MCE) solution. I attempted to dissolve solid cholesterol in ethanol (20 mg/mL), but it took an extremely long time to dissolve and remained incomplete. Moreover, when I added it to the cell culture medium (1:1000 dilution), white precipitate formed immediately and would not redissolve. I also tried sonication to assist dissolution, but the cholesterol adhered to the tube walls when mixed with the medium, still failing to dissolve properly.
The most suitable solvent for Cholesterol stock solution without using solubilizing agents is currently ethanol. A concentration of 20 mg/mL is close to the solubility limit and may be difficult to dissolve fully.
It is generally recommended to try ultrasonic-assisted dissolution first.
Since cholesterol itself is hydrophobic, at high concentrations it tends to precipitate out in culture medium. You may try increasing the ethanol proportion within the range tolerated by the cells.
If this method is ineffective, consider directly purchasing our Cholesterol (Water Soluble) (HY-N0322A, MCE) or methyl-β-cyclodextrin (HY-101461, MCE) as solubilizing agents.
The answer to this question comes from MedChemExpress (MCE) Technical Support. [email protected]
Hello Yan Yan Zhao
You may follow the protocol provided below.
1. To 5mL of Triton X-100 (in a container suitable for heating), add 500mg of cholesterol.
2. Heat the mixture gently using a hot plate or water bath while stirring continuously with a stirring bar until the cholesterol completely dissolves.
3. Slowly add 90mL of distilled water (or desired buffer) to the hot cholesterol-Triton X-100 solution while continuing to stir.
4. Stir and allow the solution to boil for 30 to 60 seconds. The solution may appear cloudy at this stage.
5. Cool the solution under running water with gentle agitation. The solution should turn clear as it cools. If the solution remains cloudy, warm it slightly while stirring until it clears.
6. Bring the solution to a final volume of 100mL with distilled water (or desired buffer).
This stock solution is generally stable for about one week at room temperature. If it becomes cloudy, warm it slightly while stirring until it clears. Depending on your needs, you may need to sterilize your stock solution after preparation (e.g., using filtration).
Please note that for cell culture applications, this stock solution will likely be too concentrated and needs to be further diluted in your chosen cell culture medium or buffer immediately before use. Determine the appropriate working concentration based on your experimental design and the sensitivity of your cells to Triton X-100. Ensure that the final concentration of Triton X-100 in your cell culture is within acceptable limits for your cells. Concentrations below 0.15 mM (approximately 0.015% v/v) have been shown not to affect cell viability, according to the article attached below.
Article Triton X-100 concentration effects on membrane permeability ...
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