I culture oral cancer cells in polystyrene plate, these cell were okay up to 24 hours, after wound has been created to analysed the migration efficiency, after 48 hours when i check again these cell were completely detached from surface of plate and morphology of cell were also changed..before experimental i check my DMEM media (10%FBS and P/S) and cells both were okay. now i don't understand what happened to these cells..could any one give me valuable suggestion to improve my experimental.
The important point is cell density on your plates at the initial point.
If your cells reached confluency and after that you wounded them - their attachment to the plastic could be distorted.
In fact, there are 2 different moments to keep in mind - attachment of the cells to plastic and adherence between the cells. If you have low density of the cells - adherence to the plastic dominates, when the cells reached confluency - cell-cell adhesion increases and at some moment may become stronger than adhesivity to the plastic... (This "critical point" strongly depend on your cell type). In such a case, you could state the cells as "thin film" and if you will wound it, it will lose a contact with underlayer plastic...
so, I would advise to check confluency, adn start a scratch assay at the confluency not more than 80-90%.
The important point is cell density on your plates at the initial point.
If your cells reached confluency and after that you wounded them - their attachment to the plastic could be distorted.
In fact, there are 2 different moments to keep in mind - attachment of the cells to plastic and adherence between the cells. If you have low density of the cells - adherence to the plastic dominates, when the cells reached confluency - cell-cell adhesion increases and at some moment may become stronger than adhesivity to the plastic... (This "critical point" strongly depend on your cell type). In such a case, you could state the cells as "thin film" and if you will wound it, it will lose a contact with underlayer plastic...
so, I would advise to check confluency, adn start a scratch assay at the confluency not more than 80-90%.
Dear Farhan
I am sending you a paper attached with this mail. Hope this will help you.
hi,
For cancer cells, you can also use 5% - 15% FBS. Also confluency matters when you start tl perform assay, meaning it should be around 75% only. Good luck
Thank you so much for kind suggestions, actually i used 90% confluence cell for the assay.
regards
Hi Farhan,
From your description, I think the detachment of the cells from the polystyrene may be due to the hydrophobic surface of the substrate as we know that polystyrene is a very strong hydrophobic molecule. To improve your experiment I suggest that the hydrophobic surface of the substrate be reduced in one of two ways:
-Some units of styrene in the polystyrene can be oxidized to have some hydroxyl or ketone or aldehyde or even an carboxylic acid groups attached to it so that the polymer become less hydrophobic or
-polystyrene can be coated with some other polymer which is known to be less hydrophobic. You can then do experiment to determine first the conditions in which the cells will not be detached from the coated substrate, this may happen at certain pH or temperature.
Regards
- Badges
- Science method