We are changing some parameters for producing high tiger lentiviruses. So we use Chloroquine as a transfection agent at the concentration 25uM for 8 hours. Than changing the media. So far we detected much higher transfection efficiency with Chloroquine than conventional CaPO4 transfection at 24 h. We use two different conditions with %2 FBS and %10 FBS. At the first day %10 FBS seemed to provide more transfection efficiency than %2 FBS. Both were higher than conventional method. At 48 hour there are still high RFP signals but cells are started to detach in both transfection but less in %2 FBS. We were harvesting and collecting media 3 times, at 48, 72, and 96h with the conventional CaPO4 transfection. But it seems it would not be efficient with Chloroquine. There are some protocols that collecting media just once 2.5 days after transfection. I am not sure if cells remain healthy by not changing the media for 2.5 days. Any recommendations would be appreciated.
In previous experiments that I did with and without chloroquine, I found the same result as you did that chloroquine-treated 293T cells prior to transfection was far superior than without chloroquine. Also, I found that collecting vector supernatant daily without chloroquine is not as abundant as a single one-time collection between 48-60 hours after initial transfection with chloroquine. For me, I would add the chloroquine, change the media about 8-10 hours later, and then leave the media on for another 40-50 hours to collect the vector at one time point (48-60 hours after initial transfection), and you will get good titers (10e6 TU/mL) for most vectors if you are titering by fluorescent protein expression.
Thanks for your answer. I have setup a new experimental design to try these harvest times. I hope I will get those titers.
I have never used chloroquine as a transfection agent, CaPhos seemed to do the job on its own (if you consider 10e10 TU/mL good titer). I used to change the media 24h post transfection, then let the cells grow undisturbed for another 48-72hr (72hr gave higher titers). I would then run the media through a 0.45um filter, spin at 28 thousand g for 1.5hrs in an ultracentrifuge, then resuspend the pellet in 200ul PBS, aliquot and store at -70C. Regarding the cell detachment, either the cholorquine is toxic to the 293T cells, or they are at high passage number. If that's the case, I'd thaw another vial and start fresh. Hope this helps.
Hello Hale,
I've never used chloroquine but I also faced the problem of detaching cells after transfections.
I performed transfections of HEK293T in OptiMem without serum, changed medium 6 h post transfection and used an increased volume of culture medium (DMEM, 10% FCS, 25 mM Hepes, high glucose). You will concentrate your virions by ultra centrifugation anyway. The higher volume will reduces cellular stress which can occur during the extended culture periods without medium change. Just think about an increase in metabolic waste or an osmotic stress due to evaporation. In addition remaining transfection reagents that might have toxic effects become more diluted.
I used a cell density of approx. 6x106 HEK293T cells per 100 mm dish (Nunclon delta surface) and 18 ml of upper mentioned culture medium. Harvesting after 60-72 h gave best results.
k r
Nicolas
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