We have the iPSC feederfree an wait until they are about 80% confluent.

Then we add the lentivirus into iPSC medium (mTESR or E8) and replace with fresh medium after 4 hours (no centrifugation, not transduction supplements). You can splitt the iPSC next day. If you have a selction marker such as puromycin start the selection after 24hours, but at 5-10 times lower levels than in 293T cells otherwise you kill all.

Hi Walther

thank you for your help about my ips transducing problem, but we don't have neither E8 nor mTESR. We culture our ips with DMEM-F12 and KSR containing L-Glu, non essential aa, and bFGF, on MEF as feeder.

Do you add lentivirus while ips is on the MEF or make them feeder free before transduction? If yes, how?, because we don’t have rock inhibitor too!

I wonder if I could make them feeder free on 2% gelatin, and by increasing bFGF up to 80ng/ml, then add lentivirus with polybrene(1µg/ml). Could you please guide me by this situation?

transduction on MEFs is inefficent but works. Can you select for transduced cells?

Do you intend to do clonal selection after transduction?

Then I would not worry to much about efficiancy and go ahead on MEFs. I dont think that feederfree with your medium and gelatine works(they will differentiate or die)!!!

I tried transduction on MEF, but most of the viruses had been taken by MEF and we had a strong background. If i could transduce them, I can pick them up by loop system, or stabilize them through puromycine selection.

Another point! Can I transduce them in the falcon or non adherent plate for about 12 hours, then transfer them on the MEF?

Hi,

1. Prepare the MEF conditioned medium

2. Optimise level of Acivinin A in MEF conditioned medium

2. Transfer iPS cells onto plate coated with Matrigel with collagenase IV

3. Culture iPS cells in MEF conditioned medium supplemeted with Activinin A

4. Add lenitviral particles in to the iPS cells cultured in the MEF conditioned medium

Can I transduce them in the falcon or non adherent plate for about 12 hours, then transfer them on the MEF?

No. The embryoid bodies will be formed.

Good luck!

HI Justyna. thank you

We don't have Matrigel unfortunately, and I wanted to know if I could replace it with 2% geltin or Laminin-poly-L-Lysine, though I wonder if it would work. if not I must supply matrigel ineluctable?. 

And, how should I optimize activine A in MEF conditioned medium?

No. The embryoid bodies will be formed.

Would it be good if transduction and EB formation happen at the same time?