From my experience with HEK293 cells they are not especially adherent, typically our lab treats them for 4-5 minutes with a solution of 0.5mM EDTA in PBS to detach them. If the cells are "peeling" away this suggests that you are allowing the cells to form a monolayer, which introduces competition for nutrients and growth factors promoting cell mutations. In the future those cells should be split at ≤80% confluence. If this is the case you may need to thaw out a liquid N2 stock and start over. Some other possibilities are (1) that the rapid addition of PBS directly to the surface of the plate may be creating significant shear stress disrupting them, in which case slow addition of PBS to the side of the dish would remedy this. (2) The cells are infected (3) These are highly passaged cells that are not acting correctly.

Also when I seed 10cm dishes, I've used anywhere from 5x10^5 to 4x10^6 without issues. Hope this helps.

Dear Lance,

This sounds like the typical behavior of HEK293 cells. These are, as you suspected, not as strongly adherent as for example HeLA or HN10. You might have noticed that they are of more spherical shape instead of growing flat on the dish surface.

Just handle them very carefully during wasing steps, so they don't come off before they are actually supposed to. To detach them from the plate, many people add 1 ml trypsin to a 10 cm Petri dish, rinse and pipet up and down a bit. Others even do it without trypzination, just shaking them off the plate.

Best, Laura

Bryan and Laura have tried to answer. One needs to know that there are a number of HEK293 cell lines  for different purposes. To transfect cells at very high efficiency there are different kinds of cells. For routine purpose, there are poorly adherent HEK-293 S  (where S stands for suspension) of cells. These cells can be adopted to grow differently using different culture conditions. Therefore, if firm attachment of cells is needed, these cells can be attached to Costar coated plates (this may reduce transfection efficiency).

Thanks to all of you for your advice. I've read some more and see that the cells we're using tend to detach from the plates at room temperature, so I'm going to try harvesting without a wash step using some protease inhibitors. Barring that, I'll take Laura's advice. 

Thanks again to each of you!

Hi!!

Hope your problem might have solved till now. In case if it is not, let me tell you,I also work with HEK 293 cells and use same method as yours. Therefore, I guess this can be because of your washing style with PBS. I usually prefer to pipette out PBS on the walls of petri dish or along the sides. Because the force by which we pipette out PBS is too hard for cell that make them detach from surface. So, you must be gentle while pipetting out the solution. I usually do it with   2ml or 5ml  pipettes.

Thank you to everyone who's answered this question.

To sum up my experience with HEK293Ts since answering this question in case someone else is looking:

1) HEKs aren't incredibly adherent, they do come off the plate easily. Ultimately, be gentle with them. Even a stream of liquid coming out of your pipette with too much force can dislodge them.

To harvest cells post-transfection, remove media, and trypsinize the cells, add media+FBS, then spin down the cells, wash in PBS, spin down again, remove PBS and lyse in your preferred buffer. OR, remove media, and scrape them in PBS+protease inhibitors, spin them down, wash them again in PBS, then spin down and lyse

2) If doing microscopy, incubate your (cleaned!) glass slides in a solution of Poly-L-Lysine for approx 2 hours at RT, or for an hour at 37 degrees. This works wonders in terms of keeping them attached during various wash steps, though continue to be gentle with them when adding any solutions.

Lance Doucette how much HEK-293T cells you used per 6 well plate?

Komal Saleem usually i plate about 200,000 for a transient transfection

The HEK293T and HEK293 cells that I've worked with:

1) adhere to TC-polystyrene plates quite well but less strongly than some other lines (3T3, U2OS, Hela). Generally they are polygonal, but less flattened than many other cell lines, some cells may be more rounded. However, if you shake them a bit with the media they grow in (DMEM or others + 5-10%FBS) or remove/put the media back, they will be fine. Though, excessive media changes can cause cells to detach at some places.

2) They unconditionally require calcium to stay attached to TC-plates.

If you wash them by PBS (calcium-free), they will start to detach massively which can be a problem (and surprising at first).

Many common cell lines will not detach under PBS but actually you may still notice some changes in morphology after only a few minutes. Washing by PBS+calcium, magnesium (0.9 & 0.5 mM) will not detach HEK293 cells. (Generally, it is better to wash any cells with Ca/Mg-containg solutions if you just want to change the growth/assay media to another one after that, so that changes in morphology are minimal).

In fact, this detachment by PBS can be used if you want to propagate them without trypsin: just leave them under PBS for 5-10 min at 37C and they will all detach; then they can be resuspended and passaged.

3) Poly-lysine coated TC-plates greatly promote their adherence.

(e.g. cover the surface with 0.1 mg/ml poly-K solution, wait 5-10 min, remove, wash with water, remove).

This can be used in experiments where you need to wash/replace the media or shake them multiple times.

If poly-lysine coated TC-plates are used, then HEK293 cells can be washed (at least if calcium is present) as any other regular cell lines without any cells detaching. By the way, they will also grow a little bit faster and distribute noticeably more evenly on poly-K coated plates (and the majority will look more uniformly polygonal).