A549 cells were attempted to be drug-resistant by exposure to drug treatments for a total of 5 months. After the applications, the cells were tried to be removed by trypsinization but could not be removed. Below are the trypsinization processes tried.
Trypsinization was performed classically by incubating 0.25% 1x trypsin for 3 min in a 37 degree incubator containing 5% CO2. But the cells could not be removed.
When the cells could not be removed, the process was tried again for 5 min and 10 min.
The amount of trypsin was increased (3 ml in a T25 flask) and incubated for 5 and 10 minutes, respectively.
A new, unused trypsin was applied to cells that still did not removed. The newly applied trypsin was also tried to be activated with high volume, 5 and 10 min incubator applications.
The trypsin concentration was increased. Trypsin at 10X concentration was applied in 2 ml volume. It was kept for 5 and 10 min, but the cells could not be removed again.
Finally, the trypsin in the cells was inactivated and the cells were taken back to fresh medium.
Hello Nazlı Sariye Tayyan
You may try the following protocol.
After removal of culture medium from the culture vessel, wash the cells with PBS (without Ca2+ / Mg2+) as the cells require Mg2+ or Ca2+ for attachment to substrates (in order to remove all traces of serum). Aspirate PBS and give a brief rinse with pre-warmed trypsin (few secs). Then add pre-warmed trypsin or trypsin/EDTA solution to completely cover the monolayer of cells and place in 37 °C incubator for ~3 minutes. Do not Incubate cells with too high a trypsin concentration for too long a period as this will damage cell membrane and kill the cells. If less than 90% of cells are detached, incubate the culture vessel for another 2 minutes and observe the cells under microscope for every 30 seconds. Once cells appear detached, add pre-warmed complete growth media to inactivate trypsin. Gently disperse the medium by pipetting over the cell layer surface several times to ensure the recovery of most of the cells.
There are several factors that could cause difficulty in detaching the cells from the substratum such as cell type, population density, serum concentration in the growth medium and potency of trypsin.
Hope this helps!
Good Luck!
You can also try washing your cells with a PBS/EDTA mixture to help prime the cells for detachment. Don´t be afraid to tap the flask hard to encourage the cells to detach as well once they´ve had trypsin on them.
Wash the cells with HBSS twice before trypsin and use harsh mechanical force on the flask as well.
Good Luck
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