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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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S2 cells
Posted by: WashU (IP Hidden, New member, 1)
Date: June 15, 2005 01:35PM
I am preparing for expressing hiv full-length gp120 protein . I encountered difficulty problem. I use PMT/Bip/V5-his vector, S2 cells were co-transfected with the pCoBlast vector (Invitrogen) according to the protocol supplied by the manufacturer(make use of lipofection). After screening S2 cell two time (using the antibiotic Blasticidin). I founded S2 cell are dead, as well as the control of S2 cell. Can anyone share any experience on S2 transfection? Why did S2 appear the situation?
Re: S2 cells
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 16, 2005 01:44PM
I would like to help you, but need you to answer a few things first. Are you transfecting the cells with a plasmid for resistance to Blasticidin? If so poor transfection efficency could explain why the cells died upon exposure to the antibiotic. What is your control? Are the control cells supposed to die? Are you using the minimum inhibitory concentration of Blasticidin? Do the cells appear healthy following transfections? Some reagents can cause toxicity with certain cell lines.
Re: S2 cells
Posted by: willG (IP Hidden, New member, 1)
Date: June 28, 2005 10:17PM
you shoud check whether there is S2 transfection reagent at altogen biosystems at www.altogen.com - this company have like 30 transfection reagents for many cell lines - we have successfully used their reagents for HepG2 and LNCap cells siRNA transfection (those are very very hard to transfect)
good luck WG
Re: S2 cells
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: June 29, 2005 05:55PM
I think most people prefer one-for-all reagent, altogen sells single reagent for individual cell line (one-to-one). In another word, if you have 3 different cell lines in your hands, you have to buy 3 different reagents from them. Their price are not cheap (most 0.5 mL pack) and this is not economic for most labs either (but huge profit for this company?). NEED TO THINK TWICE..................
Re: S2 cells
Posted by: 3'-UTR (IP Hidden, Unregistered user, )
Date: June 29, 2005 06:05PM
It is possible to use one reagent for these cell lines. My lab used Gencarrier-1 on plasmid DNA transfection of Drosophila S2, and human HepG2, Lncap, PC-3, DU-145 prostate lines with good success. Chemically synthesized siRNA should give higher efficiency than plasmid DNA.
Re: S2 cells
Posted by: thomas (IP Hidden, New member, 5)
Date: July 3, 2005 01:23PM
well, 5'-ATCG, you're absolutely wrong - I and many many other scientists would prefer the reagent which actually WORKS! It sound like you don't know this, but most of the transfection reagents out there doesn't work as they are claimed to do, so its a huge waste of money.
Re: S2 cells
Posted by: Johnn (IP Hidden, New member, 1)
Date: July 13, 2005 07:59PM
we used Gencarrier-1 and Gencarrier-2 => Extremely toxic to cells !
Re: S2 cells
Posted by: thomas. (IP Hidden, Unregistered user, )
Date: July 14, 2005 04:43PM
Johnn,
What cells you're working on? In my hands, both Gencarrier-1 and gencarrier-2 works perfectly on drosophila S2, COS, 293, 3T3, CHO......I did not noticed any cell loss using both reagents in contrast to lipo2000 which showed high toxicity for longer exposure.
Re: S2 cells
Posted by: Johnn. (IP Hidden, Unregistered user, )
Date: July 14, 2005 04:58PM
we used Altogen's transfection reagents ==> most unstable, toxic, pricy reagents on market, what they're thinking? we used Lipofectamine2000CD => Extremely toxic to cells! we used Fugene-6 => Extremely toxic to cells! we used Effectene => Extremely toxic to cells! we used Superfect => Extremely toxic to cells! we used GeneJuice => Extremely toxic to cells! we used jetPET => Extremely toxic to cells! we used ESCORT => Extremely toxic to cells!
Re: S2 cells
Posted by: hutman (IP Hidden, New member, 5)
Date: February 18, 2006 09:18AM
I have better experience with electroporation with all these cells. I use nucleofection with GTporator (GENTREND) solution which gives about 80% tranfection efficiency, but half price of Amaxa. It is even better for siRNA delivery.
Hutman
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