Hi Uchurappa, How are you preparing your lentiviruses? I usually filter the viral supernatant (293T cell supernatant) with 0.45um filters before I directly infect or concentrate. If you can't find the source of contamination, i would perform a small inoculation of all of your reagents (polybrene, media, lipofectamine, calcium chloride reagents, etc...) in culture media and leave for several days at 37C CO2.  your source may be your transfection reagents.

Thank you Mahmood, in parallel  I am culturing the uninfected cells but those are fine. I am not filtering the virus but virus storing at -80 and polybrene at -20 so virus and polybrene should not be a problem as such. 

I dont know exactly but I am thinking that after infecting with virus cells might loosing the immunity so those are very prone to contaminate or sth else? 

Uchurappa,

I would still try to inoculate the polybrene and a bit of the virus just incase something got in before you stored at -80 or -20. Sometimes these things happen. It seems that maybe you purchased commercial lentivirus? in that case it probably should be fine, but if you make your virus in the lab, there are many steps that can potentially contaminate your viral preps. 

Also, what are you exactly seeing in your contaminated culture? (turbid supernatant, yeast, bacteria?) 

Mahmood,

I always get turbid kind of contamination and we can easily detect it in the  MTT assay, it spoils the whole MTT results. Cells has contamination we can detect in the MTT assay so we considered that MTT is the gold standard assay to detect any contamination.

I am trying to follow what ever you suggested above, lets see.

Thanks.