when to add polybrene for lentivirus transduction? - (Jan/17/2015 )

Hi, 

I have produced lentivirus. To test the produced LV, I did two experiments. LC expresses constitutive GFP. 

1. The LV fresh from 293T supernatant were added to target cells without polybrene. The cells showed GFP in 24 hours. The LV worked in this case. 

2. In the second case, I froze LV at -80C. After a few hours, I added LV to cells with polybrene (8ug/ml). I first added polybrene to media on the cells, then added LV to this media. There was no GFP expression in 24 hours. 

My questions are: 

A. is my protocol to add LV with polybrene okay? Should polybrene be added to LV first and then to the cells? 

B. Is there a special technique to freeze the LV? 

Thanks, 

John. 

1. I add polybrene to the tube containing virus and media, remove the existing media from cells, and replace it with p+lv media.

2. No. Just take the sup (or concentrate) and leave the tube in -80. You can snap freeze it if you want as some recommend, but I don't think it necessary (check some protocols).