Hey!
I just started working with NIH3T3 Cells and this week I conducted my second plasmid DNA transfection with lipofectamine2000 in this cell line.
Unlike the first time, which worked perfectly, 3 hours into the transfection I noticed my cells started dying and the survivors nuclei started looking like crescents or really thin donuts or onion rings and more like epithelial cells rather than fibroblasts. The transfection didn't work.
I work a lot with SN4741 cells and I used my SN4741 cell media to culture the 3T3 cells as all the others in my group are doing the same with no deleterious effects on the cells.
The main difference between our SN media and the 3T3 media recommended by ATCC is the serum (we use FBS and not BCS) and we supplement the media with extra L-Glutamine and Glucose.
Can someone tell me if this morphology is to be expected or if it is otherwise abnormal?
Hello, I worked with NIH3T3 cell line for 3.5 years. It is very difficult to culture because it is easy to change the morphology if cell condition is not good.
I grow cell in DMEM+10%FBS+1%P/S (antibiotic).
Currently invitrogen company sell lipofectamin LTX and lipofectamin 3000. You should use these new products because it will reduce cell toxicity. You will not observe the cell morphology change or cell death.
The below is the image of healthy NIH3T3: http://upload.wikimedia.org/wikipedia/commons/5/5a/NIH_3T3.jpg
If you have any questions, let me know. It is just difficult in the begining. Later on, you will find out it is easy to handle with NIH3T3.
Hi,
Maybe you should also take care of the quality/purity of your plasmid DNA, e. g. by using an endotoxin-free plasmid kit when preparation from bacteria. The amount of DNA might also be a critical factor. Also, try out transfection without serum, if you did not so. Good luck.
- Badges
- Science method
- Similar topics
- Biological Science
- General Biochemistry
- Biochemical Techniques
- Cytology