I have increased the FBS content to 20%, after 3-4 days of observation the MDA-MB-231 cells are not attaching to my flasks. I'm using DMEM high glucose media for this. Can I have some suggestions please.
Hello Naomi Sanjana S
MDA-MB-231 should adhere to the substratum within 24 hours.
Below are the suggestions.
1. You should thaw the cells quickly. There should still be a few ice crystals left after thawing. Thawing cells quickly prevents recrystallization both outside and inside the cells, as small crystals may re-form if thawing is too slow, causing more ice injury.
2. Use pre-warmed media. Moving from a sub-zero environment straight to room temperature will put cells under a lot of stress. So, it is important to make sure that your cells are put into pre-warmed media right after rapid thawing to help dilute the toxic DMSO present in the freezing media.
3. Do not let cells sit in DMSO too long because DMSO has a damaging effect on the cells due to which the expression of adhesion molecules known to attach cells to the surface of the culture vessel may decrease and cells may fail to attach to the substratum after thawing.
4. Do not use heat-inactivated FBS unless specified. Heat-inactivating the FBS has been shown to inactivate complement proteins and can affect other factors like vitamins and amino acids (among others) that are present in the serum. Some studies have shown that heat-inactivated FBS will negatively affect the attachment of cells to the culture vessel.
5. Avoid contamination during the thawing process as contamination is also one of the factors which can affect cell adherence to the culture vessel.
6. Avoid using such a high concentration of FBS (20%) in the media as it can have a negative effect on cell growth.
Best.
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