Lipo has toxicity- use no more than 1.2 uL per well if a 24well plate.

first of all test your plasmid without lipo on 90% confluent cells (toxicity of your extracted plasmid )

4h after transfection change with fresh medium. I would not recommend washing your cells with PBS or withdrawing serum during tranfection steps (except for forming transfection complexes step ).

Your plasmid may encode something toxic- use a GFP plasmid as a control to assess both cell survival and transfection efficiency.

cell passage is so important.

BEst

There is no antibiotic in any of the media during the transfection?

You can boost your transfection efficiency by tweaking certain parameters like cell density, DNA to lipid ratio and forward vs. reverse attachment. You should also try keeping cells in log growth before transfection (never allowing them to become confluent).

There is no antibiotic; the cells are grown in just plain DMEM with 10% FBS. I‘ve seen this happen for several plasmids, so I know it’s not a plasmid problem either. Also, the cells were still in log growth at the time of transfection. What could be happening?

Your DNA:lipid ratios sound ok, what passages were the cells during transfection? Do you have any data with lower DNA input? What is your confluency like after tranfection? In my experiences even robust cells such as HeLa cells start to die off in 2-3 days when fully confluent. How many cells are dying? A small amount is permissable. How did you measure cell death, via microscopy, LDH or flow?

Can we rule out endotoxins?

https://www.sigmaaldrich.com/life-science/stem-cell-biology/3d-stem-cell-culture/learning-center/what-is-endotoxin.html

Looking under a traditional light microscope 6 hours post-transfection when I change the media, not many cells are dying - less than 5-10%.

Same case when checked prior to fixation. Under confocal microscopy, the transfected cells, which represented less than 10% of all cells, were all dead or dying - they all had apoptopodia or had already broken into apoptotic bodies. I don't have any data with lower DNA input... should I try that? As for endotoxins, we use a midi-prep system with an endotoxin removal wash, and plasmids are prepared on a lab bench with standard sterile procedures, clean gloves, ultra pure water, etc. Thanks so much for the advice - any idea, based on this, on what could be happening?

From my experience i also observed 5-10% dead cells after 4-6 hour incubation so this may not be so usual. Transfection can be cytotoxic with varying DNA to lipid combinations, i.e. 1ug to 1ul lipid is not the same as 4ug to 4ul despite being a 1:1 ratio. What is your transfection efficiency?

Transfection efficiency was about 5-10%. All transfected cells were apoptotic, but untransfected cells were not affected. I prepared lipid complexes for 4 wells at a time. I started by combining 4 μg DNA with 8 μL P3000 and 200 μL Opti-MEM in one tube. Then I combined 4 μL Lipofectamine 3000 and 200 μL Opti-MEM in a separate tube and let sit for 5 minutes. After that, I removed the diluted Lipo 3000 and combined it with the diluted DNA/P3000 for a total volume of ~400 μL and let sit for 15 minutes. I added 100 μL of that mixture to each well in the 24-well plate dropwise. Is there anything wrong with the order I’m doing things in?

In that case you may need to perform transfection optimization.

A good starting point would be to rule out mycoplasma contamination- i have seen the effects of myco+ cells on transfection efficiency. It is a very subtle contamination. Once you rule that out, you can try:

5.3.3 Transfection optimisation or pg 160/318 from:

http://epublications.bond.edu.au/cgi/viewcontent.cgi?article=1195&context=theses

I think your DNA concentrations are too high, with 4ug DNA i observed high cytotoxicity (figure 5.10).

Also if we check the lifetech website for L3K, we see that it is validated in HEK cell line with high efficiency rating.

https://www.thermofisher.com/tr/en/home/brands/product-brand/lipofectamine/lipofectamine-3000.html#validated

1ug of DNA for 1 well of a 24 well plate sounds like quite a lot. 90% confluency also seems rather high for good transfection efficiency, especially if you need to leave them for an additional 48 hours post transfection. I would try transfecting them when they're not quite so confluent, around 70-80%, or even as low as 50-60% since you need to leave them for a couple of days. You should be able to get decent transfection with about 200ng of DNA in a 24 well plate depending on the construct being used. Changing the media 1-4 hours after transfection should also help. If you're still having trouble with toxicity, try times on the shorter side. An additional thing to try would be to coat your surface with fibronectin, collagen or PDL/PLL so that your Hek cells have an easier time staying attached while you're exposing them to toxic substances, including the PFA you use to fix them. Be sure you're cells are not at too high of a passage number, once they get above 25-30, they usually get pretty unreliable and start to break down.

293T are quite resistant cells and should not die 48h post transfection.

70-90% confluency is good to start with and the higher the confluency the more reistance to transfection is expected.

However, if the confluency is too high at the time of transfection you might get low efficiency of transfection but otherwise you should get 90--100% efficiency with a control GFP plasmid DNA.

With Lipofectamine reagents, it is important to change the medium 6h post transfection especially for sensitive cells.

500 ng is recommended for a 24 well plate but 2x should not be a problem.

I see two possibilities here: your cells are in bad shape already before transfection or your plasmids are toxic but in that case, the non transfected cells should be OK