suspencion cell - electroporation with BTX830 apparatus - TROUBLE WİTH SUSPENSİON CELLS (Nov/02/2007 )

Hİ EVERYONE,

I am trying to transfect my suspencion cell lines with lipid based agents but no success. SO, i will try electroporation. has anyone tried BTX830 HARWARD APPARATUS. i have flourescein labelled siRNA siGLO, PC GAPDH and G6PDH siRNAs and Non-targeting as NC besides my target genes. any one know about CCRF-CEM, HL-60, K-562 transfection..

thanx

burcin tezcanli

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I don't know if you are locked in to the electroporator or not, but I just found a transfection reagent that works great on RPMI 8226 human myeloma cells. HiPerFect from Qiagen. After running an optimization plate I found I had great viability compared to my untreated controls and at 24h I had >90% transfection efficiency as measured by flow cytometry and Alexafluor488 conjugated siRNA. I plan to repeat the experiment next week so I will let you know. The insert for the reagent mentions K-562 cells as a targe cell line for their suspension cell line protocol.

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thanx alot, i will be pleased to hear your results.. hope that works

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Binding/internalization is not an indication of a successful transfection. Any cationic agent can facilitate uptake, but not all work as well in cytoplasmic delivery. Let us know the knockdown rate, coz thats what counts. Thanks.

K562 is not a typical suspension line and is more transfectable than the rest suspension cell types. Its more like something in between suspension and adherent cells.

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Although there are a lot more factors than uptake alone there is also the old concept of "you have to eat before you can digest". As I said before in my hands the system I mentioned worked well to get the oligo into a cell line that I had previously been unable to see any uptake or even surface binding with. I plan on looking at control gene knockdown next week.

P.S. if you talk to qiagen they might be able to hook you up with a sample of their reagent

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