Hello,
Does anyone sucessed to make THP1 cell transfection efficiency?
I am currently trying to tranfect THP1 adherent cells (PMA differentiated for 10 hrs) with specific vendor protocol. But it resulted a big cell death and also poor tranfection yield.
I followed a protocol from life technologies,Lipofectamine LTX Plus reagent as well Lipofectamine 3000 reagent. Unfortunatley after overnight incubation i observed a big perecentage of cells detached and floating/ only few cells transfected.
My transfection contents
1.Optimem Medium
2.Lipofectamine reagent
3.GFP' Plasmid DNA (1-5 µg)
4.P300 reagent or PLUS reagent
My experiment requires Adherent cells and 60% transfection maximum.
Here , I am expecting some optimised protocols or your experienced suggestions for trouble shooting.
I am willing to follow all your protocols , suggestions, and opinions.
Thanks in advance,
sriki
I had this exact issue a few years ago and ended up moving to a more transfection-amenable cell line, but I called Life Tech customer service at the time and got some advice that may help you.
1. Be absolutely sure that your plasmid prep is as endotoxin-free as possible
2. Try using less DNA
3. (Most important) Wash the cells and add fresh medium after 4 hours. This should be sufficient time for a good portion of the cells to take up the plasmid and will remove the toxic lipids from the LTX reagent, even though it's supposed to be reduced toxicity. THP1 are extremely sensitive to any perturbation, and the lipids are definitely enough to wreak havoc.
Thanks Jordan,
OK, i will try this and let you know the effect.
thanks
wash, wash, wash. as jordan above said, the real secret in transfecting pma-difrentiated thp1 is to wash the hell out of that lipofectamine reagent and add very fresh medium, preferrably fresher than the one you used to maintain the cell line. check ph of both media and of pbs 1x wash buffer. recheck if maintained for longer periods of time (+2 weeks).
Hi Shrikanth,
I have used THP1 suspension cell in the past. I used Amaxa nucleofector (electroporation) and transfected siRNA using T-001 program. Transfection efficiency was roughly 60% and cell viability was quite good. Other people in my lab have also transfected plasmid DNA into THP1 using the same method.
Hope this helps!
Good luck.
Prahlad, what is your outgrowth protocol? Meaning, what steps do you do after you transfect to care for the cells?
After electroporating the cells (2 million cells in 100ul of serum and antibiotic free media), I leave them for 5-10 mins at room temperature so that cell can recover from the shock. Then I add 0.9 ml of the media without antibiotic and re-suspend the cells well using the plastic transfer pipette and transfer them to the 12-well plate containing antibiotic free media and incubate at 37 C for 24-48 hours before analyzing.
I recommend testing the transfection reagent DreamFect which has been successfully used for THP-1 cells.
Here you can find example of publications:
http://www.ncbi.nlm.nih.gov/pubmed/24517546
http://www.ncbi.nlm.nih.gov/pubmed/22140256
for your convenience I have attached a specific protocol for suspension cells.
Otherwise if you differentiate them into adherent cells then I suggest testing Magnetofection technology (see example of publication):
http://www.ncbi.nlm.nih.gov/pubmed/22087003
do not hesitate to contact me if you have any questions
Best regards, Olivier
Hi,
I am having actually the same problems, though I am using Polyplus Jetpei Macrophages kit (as well recommended by some researchers here besides the LTX Lipofectamine). Still I did not try another protocols like removal of the transfection reagent after four hours.
Do you have any follow up comments on your experiments? Did you make it work? I begin to be a bit hopeless with these reagents.
Thanks in advance.
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