Hello,
I have been trying to use the CRISPR-Cas9 system in u373 cells using PEI chemical for transfection. I begin procedure by seeding the cells in a 6 well plate at a 1x10^5 cells/mL density, letting them grow overnight and begining transfection the following day. I use the PEI chemical in a 3:1 ratio, changing the medium after 24h of transfection. However, after those 24h a lot of the cells are floating, which I presume is due to the PEI chemical or the transfection procedure as well. Is there any way of preventing this cell death and optimizing the tranfection procedure with the PEI chemical?
Thank you.
Hi. Would you mind giving little more details of your protocol. Like media, FBS, incubation time of PEI-DNA complex etc. You will surely benefit from adding little more cells, something like 5E5 cells per well of 6-well plate.
Hello,
I would suggest you to use PEI at a concentration of 1mg/ml and also change media after 4 hours. PEI is a polymer and toxic at higher concentration and also if you are using high molecular weight PEI i.e. 25000 kDa then you must dilute it to a concentration of 1 mg/ml.
Please try this and change media after 4 hours of transfection.
Also I will recommend you to grow 7*10^4 cells per well, it will give you better transfection results and studying transfection efficiency later.
Syed A Ali
Hello,
The media that I'm using is MEM suplemmented with the folowing: 10% (vol/vol) fetal bovine serum (FBS), 1% (wt/vol) glutamine, 1% (wt/vol) nonessential amino acids, 1% (wt/vol) penicillin/streptomycin and 1% (wt/vol).
Once they have around 70% of confluence I dilute 1 µg plasmid DNA in 100 µl of serum and antibiotic-free cell culture medium. I mix the PEI with the DNA and incubate at room-temperature (RT) for 10 min. I then add antibiotic-free cell culture media (600 µl) to the transfection mix, and the total volume is added to the cells. I incubate the cells for 2h with the transfection mix and after those 2h I add 2ml of antibiotic-free media and incubate at 37ºC for 24h.
Richa Pandey Thank you! I will increase the cell density for the next experiments.
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