transfection of RAW 264.7 - (Mar/31/2007 )

I tried transient transfection of RAW 264.7 to perform a luciferase reporter assay using Promega kit.
I used Tfx20 for transfection using 0.8ug DNA in 3:1 charge ratio to Tfx. I did not managed to detect a signal when performing the assay. The DNA was successfully transfected in HeLa cells. I have tWo probable problems why this happened:

1. The transfection did not worked
2. Luciferase degraded due to increased temp during lysis at 37 for 30 mins (the shaking incubator was accidentally switched on)

My questions are:
1. Did someone ever successfully transfected this cell line using Tfx20 (Tfx50 was used and the difference is only in the ratio of lipid used).

2. Does anyone knows the stability of luciferase after lysis. Promega states thet it is stable for at least 6 hrs at 22. Please does anyone knows whether it is stable at 37? The plasmid used was pGL3E.

Thanks.

---

Raw cells are difficult to transfection with chemical (lipid and CaPi) methods. You are luck to get 1% transfected under optimal condition.

Txf20 is not the best lipid formulation availabe.

Your best bet is electroporation or lentiviral vectors. Very high dosages of adenovirus may also work.

5 Min room temp lysis is sufficient to break down your cells.

---

Thanks for reply. I also think that 24 hours might be enough to perform the reporter assay as the cells grow very fast and I noticed that after 48hrs, in some wells the medium became acidic. There was no bacterial contamination but I think that the cells might had already started deteriorating and so also the luciferase.

what are your suggestions, please?

---

I would chose to either do the assay at 36 hr or change medium after 24 hrs, then do the assay at 48hrs. 24 hr post transfection maybe too early for some cells, but I have seen in other cases 24hr is too late, especially when toxicity is high. You may need to test it out according your particular case.

---