Here is my problem: The day before transfection, I seed the HEK293FT cells into 100mm dish(cell density 4*10^6 cells), the next day before I preform the transfection, cell confluency like 90%, and I change the medium (warm the medium before). When I did that, the cells did not detach immdiately, but after I move the dish into incubator, and wait for the plasmid package, the cells become detach from the edge and almost the whole cell layer become detach. My concern is can I re-seed these cells by pipette? Can these work? Will it effect the transfection efficiency or the lentivirus production?
I had the pretty same condition like you, HEK293LTV cells got a shrink morphology after media changing, and I thought they died. But after 24 hours, they were in a good condition. Changing of their dish (from Flasks into 10-cm dish) may cause this problem. But you can harvest all your cell-containing media into a 15-cm falcon and centrifuge it for 5 min in 1250-1500 RPM to get cell pellet and then culture this pellet into new dish. Hope this helps!
Fatemeh Eslamizadeh I really appreciate your suggestion. I encountered the same issue, I observed that after several hours (3 or 4hours) the detach cells became adhered again, but had some folded layer. And when I need to change the media again and I used pipette to detach all cells untill they became single cells. Maybe I should centrifuge to remove the dead cells like you do. The plasmid contains fluorescence signal, so I will check by microscope next day. Hopefully they were already transfected.
probably they will re adhere if you put them in a new plate at a lower density (and after separating the agregates by flushing up and down) but I am not sure that the transfected cells will reatach... to much work for an unsure result... HEK-FT are suppose to grow in suspension (F) and they adhere really poorly (you can improve adhesion by coating your plates with poly L lysine (or buy coated plate) but I would rather shift to HEK-T cells; they adhere much better on plastic (and even better on coated plates)
You might want to try coating your dishes with with 1 mg/ml gelatin for about 30 minutes, prior to seeding. I was taught to always do this for transfection in grad school. In my experience HEK293FT will adhere well by this method, so long as you are gentle.
Coating with poly L lysine (as Didier suggested) or Collagen I may also work fine, just be sure to report what you use as cells can react differently to matrix proteins.
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