H9 Cell transfection - H9 cell transfection protocol (Sep/19/2008 )
I am now transfecting EGFP plasmid into Human T cell line-H9. Following the Lipofectamine 2000 protocol of suspension cell transfection, I failed to transfect H9. During transfection, antibiotics was free from the medium. Worrying about contamination, I add antibiotics into the medium 24 hours after transfection. Is it ok?
After transfection, a lot of cells deaded in 24-48 hours. Today, I do see some green flurescence in the culture. But co-worker told me it is not the positive cell. I am confused that is there any other factor can cause green flurescence in the culture???
background of plate or medium?
I am going to transfect a second time next week. Could you provide me a protocol or some experience on how to transfect H9?
Thank you very much!
After transfection, a lot of cells deaded in 24-48 hours. Today, I do see some green flurescence in the culture. But co-worker told me it is not the positive cell. I am confused that is there any other factor can cause green flurescence in the culture???
background of plate or medium?
I am going to transfect a second time next week. Could you provide me a protocol or some experience on how to transfect H9?
Thank you very much!
Can you take and post a picture of your green cell that according to your co-worker was not a positive cell?
As far as my experience with T-cells goes, they're hard to transfect. You could (if available) try amaxa (colleagues do it with that, it's not as efficient as you'd want it to be, but not as bad as liposome-based). Do you want to create a stable cell line or just transiently transfect them? (if stable, you could do with low efficiency of transfection and upscale your exp to get enough clones to further examine).
Maybe post your protocol, LF2000 can get pretty toxic so maybe just performing the same exp with more cells can reduce toxicity? (I doubt it will increase efficiency however);
Good luck.
Yes, I am going to construct a stable expresion cell line. I did a second transfection yesterday with Lipofectamine 2000 and reagent respectively. Cells are not in good condition this morning, dead cells take up to almost 50-60 percent in 6-well plate. As you said, upscale cell munber may be helpful. The problem is how to transfect H9 successfully at first. I googled online , but no H9 transfection protocol was found. Is there anyone transfecting this cell???
Try to google HUT-78, H9 is a subclone of this cell line. Hope this helps, but T-cell lines mostly are hard to transfect. Since your goal is a stable one, you don't need to much efficiency? Can you try retroviral transduction?
Thank vairus for your suggestion. I am going to google HUT-78. In fact, I would transfect H9 with vectors and also transduct with retrovius. But retrovirus are not ready yet. These are for different approaches.