Here is the situation: I usually culture 293FT cells in 10cm dishes and coat these dishes with 0.1% gelatin overnight to enhance adherence. When I passage these cells, seeding density: 1 millon cells, wait for 3 days until the confluency reaches aroound 90%. Then wash with PBS once and dissoicate with 0.25% trypsin-EDTA in incubator for 3 minutes, add complete culture medium to stop the dissociate process. And the cells always look normol during these process.
The day before transfection, I seed 4 millon cells to the dish. Next day, the confluency was around 80%-90%. But after I renew the medium, almost of the cells detach before I add the transfection reagents. What could be causing this?
Thank you for your kind help in advance.
Feng Wang Thank you for your answer. But my struggling problem now is about the condition of HEK293FT cells. The current transfection system used in my lab is not problematic at present. How could I enhance the adhesion of HEK293FT cells? Or is there anything I should pay more attention to?
Option 1: Check whether your transfection reagent could be used for reverse transfection.
Option 2: Coat cells with different coating materials and try which one is better.
Option 3: Repeat your transfection (forward transfection), but change medium with pre-warmed medium, carefully carefully and carefully carefully. Load the fresh medium to the dish wall slowly and slowly, you can touch the dish wall with tips when loading the medium .
I did both option 1 and 3, both worked well.
In my experiene, HEK293 are quite sensitive to temperature changes and mechanical stress from adding liquids to the dish (e.g. media replacements, washing with PBS, etc.) and then easily detach. I resorted to never adding liquids directly onto the cells, but pipetting liquids slow and use via the rim of the dishes and to avoid shearing stress.
Good luck!
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