2 replies to this topic

[wangdaiming]

Hi Friends


  I am in a delima.  I have spend over one year to generate stable cell lines in LNCaP cells (prostate cancer cells).  The protein I found can enhance androrgen receptor's transactivation in transient transfection.  I introduced this factor into LNCaP cells permanently.  Western blot confirmed the stable expression of this factor in LNCaP cell. But, the effect of this factor on androgen receptor's transactivation was variable via CAT assay.  During screening, I pick up positive effect of this protein on androgen receptor's transactivation.  Next time, when I repeated CAT assay, it became negative.

I am totally confused.

Thanks for your help.

Daiming

[caro]

Hi there

Have you made clonal selection? I have seen my pool of stably transfected cells behave differently at different times, but it is eliminated by selecting clones.
I normally select clones by diluting my cells to 0,5 cells/100 microliter and distribute them in a 96 well microtiter plate 100 microliter in each well. Then you have to look for wells with more than one cell for the first couple of days. When the well is confluent you trypsinise and move the cells to a litle flask.

Caro

[caro]

oups...too fast... you disregard the wells that has more than one cell, in the first two days...