about MTS assay and plasmid transfection - (Oct/12/2006 )
Hi,
I am doing MTS assay of two breast cancer cell lines 48-hr after transfection of plasmids. For the transfection, I used 0.5ug plasmid and 2ul Lipofectamine 2000. The cells were cultured in 500ul medium (antibiotics free). Plasmid and lipofectamine 2000 mixture was added in 100ul. Everything follows the product manual. One plasmid encodes beta-glactosidase, which should do nothing to the cell. Another plasmid encodes a growth factor inhibitor, which should kill the tumor cells. However, in my experiments, both plasmids led to significant cell death. I don't know why the control plasmid didn't work as a "control". Those plasmids were collected from bacteria transformation, mini-prep, and maxi-prep. They are eluted in 10mM Tris-Cl, 0.1mM EDTA, pH8.5. Is this buffer harmful to cells? Or if the plasmids were contamined by some ethanol during the mini-prep and maxi-prep, will that kill the tumor cells?
I am really confused by my results. I would really appreciate if anyone can give me any suggestion!
I am doing MTS assay of two breast cancer cell lines 48-hr after transfection of plasmids. For the transfection, I used 0.5ug plasmid and 2ul Lipofectamine 2000. The cells were cultured in 500ul medium (antibiotics free). Plasmid and lipofectamine 2000 mixture was added in 100ul. Everything follows the product manual. One plasmid encodes beta-glactosidase, which should do nothing to the cell. Another plasmid encodes a growth factor inhibitor, which should kill the tumor cells. However, in my experiments, both plasmids led to significant cell death. I don't know why the control plasmid didn't work as a "control". Those plasmids were collected from bacteria transformation, mini-prep, and maxi-prep. They are eluted in 10mM Tris-Cl, 0.1mM EDTA, pH8.5. Is this buffer harmful to cells? Or if the plasmids were contamined by some ethanol during the mini-prep and maxi-prep, will that kill the tumor cells?
I am really confused by my results. I would really appreciate if anyone can give me any suggestion!
maybe your mock vector still express resistant factors (to select cells in ampilin, kanamycin/neomycin or others) which may perturb your cells; it will definitely not the TE buffer; cytotoxicity should be qualified by apoptosis and necrosis assays
I am doing MTS assay of two breast cancer cell lines 48-hr after transfection of plasmids. For the transfection, I used 0.5ug plasmid and 2ul Lipofectamine 2000. The cells were cultured in 500ul medium (antibiotics free). Plasmid and lipofectamine 2000 mixture was added in 100ul. Everything follows the product manual. One plasmid encodes beta-glactosidase, which should do nothing to the cell. Another plasmid encodes a growth factor inhibitor, which should kill the tumor cells. However, in my experiments, both plasmids led to significant cell death. I don't know why the control plasmid didn't work as a "control". Those plasmids were collected from bacteria transformation, mini-prep, and maxi-prep. They are eluted in 10mM Tris-Cl, 0.1mM EDTA, pH8.5. Is this buffer harmful to cells? Or if the plasmids were contamined by some ethanol during the mini-prep and maxi-prep, will that kill the tumor cells?
I am really confused by my results. I would really appreciate if anyone can give me any suggestion!
In a situation like this cross check two things-
1. plasmid preparation
2. reagents used in transfection
Excessive EDTA, carry over ethanol etc could be harmful to cells...
A both sets- control and test plasmid transfected cells are dying my guess is something common in these two sets is causing cell death.
sd