AtT20 low transfection efficiency - (Jul/20/2005 )
i used lipofectamine2000 according to manufacturer's protocol for floating cells , but encounter a vr low transfection efficiency.
when the same condition apply to my adherent cell line (NIH-3T3), the efficiency is 100 times higher!!
Anybody has a better method or approach to tackle this cell line..
We’ve used calcium phosphate, Lipofectin, Lipofectamine2000, Effectene, Superfect and Gencarrier-1 on the AtT20 D16v cells (a mouse corticotrophic cell line). We get the highest
efficiency (only 15-20%) with Gencarrier-1 (Epoch Biolabs). Selection is with 400µg/ml G418 (about 10 days to get 100+ cell colonies). We’ve screened by ligand binding, ion channel modulation, RT-PCR, and immunostaining (of living cells).
For transient transfection, cells are harvested 48-72h post-Tfx for analysis.
thanks bryan for the constructive reply.
curious question from new hand here: how do u actually check the percentage of your transfection?
another thing is that, is the heavy floating and clumping of this cell line affecting the transfection effeciecy as well ??
i heard that by increasing the percentage of serum in media will make the cell adhere. it that true and nessessary ??
Using GFP and Lac-z reporter plasmid to determine tfx efficiency.
You're right. One major trick for tfx is to avoid cell clumping as possible as you can (gently disperse to single cell suspension). This will affect a lot for tfx efficiency.
I don't think serum will help in this case. It all depends on cell type. Actually, we used serum-free medium during tfx.
Hi guys,I'm really interested in your discussion.
What I was wondering is if you are using the "original" AtT20 which grow in suspension or the adherent strain S16v-F2?