We tend to use CellBind culture-ware in our lab when transfecting HEK293 cells. They lift very easily when exposed to the toxicity of the transfection reagent.

1. Toxicity from transfection reagent. If so, the morphology may change.

2. Toxicity from the gene that you want to express. If so, the morphology may change.

3. Improper pipetting skill. HEK293 cells are easy to detach by mechanical force. Pipet reagents as careful as possible.

Have you also included negative controls?

1. NEG control without DNA but with transfection reagent?

2. NEG control without DNA and  transfection reagent?

How do the negative controls behave?

Which transfection reagent do you use/ how many cells per well?

I do have a Neg control without DNA and with transfection reagent. I use lipofectamine 3000 from life technologies. They also showed similar rates of death. I use 200,000 cells/well as these cells are relatively weak so I seed on the higher side to be safe.

This probably isn't a problem with your transfection or the gene you are expressing. HEK293 (and HEK 293-T cells which are easier to transfect) behave like this normally when they are overconfluent. Depending on the transfection reagent you should aim to transfect the cells when they are about 50-70% confluent (use medium without antibiotics for the best result). If you are finding that your cells are coming loose too easily you can use low serum medium (2-2.5%). Either replace the medium before transfection or replace it 12-24 hours after transfection. Your cell growth should slow and it should be less likely for them to peal off in a single sheet. It still pays to be gentle when handling the cells (don't knock the culture dish and pipette medium carefully down the side of the dish) as they may still wash off. If you are still finding that this only happens to certain wells and it reproducibly happens to those same wells then the protein you are expressing may be slightly toxic or may be altering cellular properties enough to have an effect on their adhesion properties.

We tend to use CellBind culture-ware in our lab when transfecting HEK293 cells. They lift very easily when exposed to the toxicity of the transfection reagent.

We would sometimes coat the plates with collagen to help the cHEK293 ells stick better.

Protocol:

Dissolve collagen 2mg/ml in 0.2% acetic acid.

Filter the collagen through 5um filter.

Filter it again through 0.45um filter, then store at 4°C.

When coating, dilute the stocked collagen 1:5 in tissue culture grade water.

Dispense diluted collagen solution to dishes and sit overnight.

2ml / 35mm Petri dish

3ml / 60mm Petri dish

5ml / 100mm Petri dish

4ml / T25 flask

12ml / T75 flask

0.5ml / 24 transwell

Remove remaining collagen solution and air-dry.

Best,

I used 0.2% gelatin to coat the dishes. (>15min in 37oC)

Seeding cell one day before (50-60%).

Wash cell with D-MEM (not PBS) before transfection. Drop the transfection solution.

Even though, I sometimes have cell detach from dish. Then I wake up another cells from LN.

Hope it help you

best

I use HEK293T cells and I perform transfection with LipoD293 (SignaGen Laboratories). It works very well for my constructs (10kb).

I seed my cells into a 6 well plate and transfected 1 ug of plasmid /well. Actually these cells are easy to detach. But no problem after 24, 48 and 72 hours

If you are seeding 12-well plates with 200,000 cells per well then I think that's your problem. Try seeding with 50000 to 100000 cells the day before transfection. I also prefer to do transfection in the morning, and change to fresh media right before I leave for the day so the cells aren't in the transfection solution all night. I transfect 293s quite frequently, and this seems to work best for me. Also, if this keeps happening even after lowering your seeding density and decreasing incubation time in Lipo3000, try lowering the amount of DNA you are using. I usually use 1ug for a 6-well dish, so I would use less for a 12-well - the cells might not like the DNA, especially if it was not prepped with an endotoxin-free method.

Thanks everybody for all the suggestions.

Danielle, do you use an endofree kit to extract your plasmids or just a normal plasmid extraction kit? And without the endo free kit your cells still survive using your method? Because if your method doesn't work for me, I am thinking of purchasing an endo free kit to try to increase the chances of cell survival. 

Vaishnavi,

I almost always use a normal plasmid extraction kit, but in the past when I tried overnight incubation in transfection media my cells almost always die, so I have switched to only leaving it on for 4-8 hours and it solved that problem for almost every transfection. I have used Lipo-only negatvie controls and they're fine, so it makes me think it's an issue with the DNA. The fact that your cells are coming off in sheets leads me to believe that your cells are way too confluent though, even if endotoxins are attributing to the cell death. 293s can handle transfection at 50-70%, so try plating them so that they will be 50-70% confluent when you do transfection, and try keeping the transfection media only on them during the day and change media before you go home for the day. I hope this solves your problem! Good luck :-)