can anyone help to me and explain in more details
I transduces mesenchymal stem cell with lentiviruse but it is not efficient...
/5 ml of concentrated lentivirus was added to each well of 6 well plat with approximately 100/000 mesenchymal stem cell and poroumycine selection was perfomed after 4 days, but was not efficient. it is important that concentrated lentivirus mixed to fbs free medium? I mix it to fbs+ medium and added to cells drop by drop
Dear Zahra Heidari
Have you added polybrene to the lentiviral concentrate and the media of transduced cells? It is commonly used to enhance transduction efficiency. Have you tested your concentrated lentivirus on other cells to make sure it is working? I performed lentiviral transduction successfully into fibroblasts, and the media I used was 10% FBS, so I think the FBS is not probably the problem.
thanks a lot/ its a good suggestion and I guess that lentivirus doesnt work because after selection puromycin there is no difference between target and control cell but 1 day after lentivirus added I see changes in morphology of target cell ... can you explain anything else that could help me ? what are these cell morphology changes for ?
It is better to provide images of cell morphology changes. In general, if you use highly concentrated lentiviral concentrate, it might be toxic to the target cells. Usually, you should use various ratios of viral concentrate/fresh media in 6 well-plate for example 0/1 1/1 1/2 1/5 1/10 1/50.
Another issue might be the time needed for the antibiotic selection, some cell lines take longer time.
Thank you very much for important points you mentioned , first I want to determine virus titration and second checked it with various ratio of viral concentrated
- Badges
- Science topic