Hi guys,
we want to use Lipofectamine for transfection of HEK cells. In the protocol, PLUS reagent is added to increase tranfsfection efficiency.
Does anyone know if it is toxic for the cells if we use a higher amount of PLUS reagent than indicated in the protocol?
cheers,
Sophie
Hi,
thanks for your answer.
no, we do not expect it to work "better" because we use more (though it would be quite awesome^^), we are just playing around with DNA concentrations (vectors) to test the method and to figure out what works best.. we were just wondering if we should have a look at different PLUS reagent amounts too, or if we could kind of neglect this.
Hello
Proposed follow the same steps of your Protocol , for the higher amount of PLUS reagent , pleas visit www.lifetechnologies.com/transfection or contact Technical Support for specialized transfection protocols (including cell-type specific advice on use of PLUS™ Reagent and antibiotics, and a protocol for vector-based RNAi).
Good Luck
http://www.lifetechnologies.com/sa/en/home/brands/product-brand/lipofectamine.html
Dear Sophie,
You can also try other reagents that do not need an extra dilution buffer such as PLUS with at least the same or even higher efficiency. HEK cells are very easy to transfect and thus DreamFect Gold (all in one) will be very good as well. You can see the publications list here: http://www.ozbiosciences.com/transfection-dna/19-dreamfect-gold-transfection-reagent.html
If you need a sample let me know at [email protected]
Good luck
Hi Sophie, if you want to easily transfect HEK with high efficiency then you should try our new ScreenFect™A reagent. We developed this product ourselves here at KIT using combinatorial chemistry and then HT screening in HEK cells. We market this product through Incella GmbH. We actually optimized for the so-called 1-step transfection method, which allows simultaneous plating and transfection of cells and saves a lot of time! We can send you a sample for testing if you want.
Hi,
thanks to all of you for your advise - we will test next week and I will give you some feedback asap.
Gary, would you mind sending me a few more information about your product?
Hi Sophie, of course. Please contact me at [email protected] and I will send you more information and arrange for some test samples to be sent if you want.
Please visit also our website www.incella.com for more information (we are currently adding more content).
best Gary
You can easily save time and lots of money if you simply transfect HEK293 with the classical calcium phosphate precipitation technique. And yes, higher PLUS concentrations are toxic, HEKs do not like lipefectamine too much. Plenty of labs use HEKs to produce Lentis. We always use calcium precipitation.
Hey guys,
we actually did the Lipofectamine transfection yesterday. However today our control cells look great while the actuall cells treated with the plasmids died over night. Any ideas?
Control cells mean what? Did you do the transfection without antibiotics, did you wash them before without antibiotics, after how long you changed the medium?
Use one of the below listed cell death controls to determine the best starting concentration
AllStars Mm/Rn Cell Death Control siRNA ( mouse/mammalian)
AllStars Hs Cell Death Control siRNA (Human).
Hi Kay, - control cells mean cells treated with Lipofectamine but without DNA.. we also did transfection w/o antibiotics but got the same results: cells detach after over night incubation.
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