Q1: Why is there no obvious mineralization after 2–3 weeks of induction?
This may be due to an excessively high cell passage number or an overly low seeding density. We recommend using cells with a passage number of <8 and initiating induction when cell confluence reaches 60%–70%.
Q2: What should I do if mineralization is weak?
This may be due to an insufficient induction period; you may extend the induction time to 21–28 days.
Q3: What should I do if cells detach in sheets during the late stages of induction?
1. Coat the culture vessels with an appropriate amount of 1% gelatin.
2. After 2 weeks of induction—to minimize the risk of osteocytes curling at the edges or detaching—you may switch from a complete medium change every 3 days to a half-volume medium change every 2 days. This helps prevent cells from floating due to the agitation caused by complete medium changes, and should be continued until abundant mineralization nodules appear.
Q4: Can the culture medium still be used if a white precipitate appears?
The white precipitate is likely caused by the low-temperature crystallization of glutamine or calcium salts; the medium can be used normally once the precipitate dissolves at 37°C. However, if significant turbidity and discolored precipitates are observed, the medium is likely contaminated or has expired and must be discarded immediately.
Q5: Why do a large number of cells die during the early stages of induction?
This may be due to the culture medium not being preheated, an abnormal pH level, or the presence of antibiotics with cytotoxic effects in the induction medium (which are more prone to damaging cells at low temperatures). We recommend preheating the culture medium to 37°C and allowing it to equilibrate to a pH of 7.2–7.4 prior to use.
Q6: Why does Alizarin Red appear yellow in some wells of the culture plate, while appearing red or purple in others?
Alizarin Red exhibits different colors under varying pH conditions, typically ranging from orange-yellow to purple. The Alizarin Red solution provided with our osteogenic induction medium has a pH of 4.2 and typically appears yellow or red. During the staining process, it is a common and normal phenomenon for the solution to appear yellow, red, or purple, as the color may shift due to changes in the local environment or the production of acidic or alkaline substances by the cell samples.