Hello! I bought this new reagent CellLight BacMan 2.0 for a protein fused to RFP to transduce my U87 glioblastoma cells and look at my moving proteins in live. I thought it was very efficient but did not actually work. In the official protocol by Invitrogen was described the working procedure on adherent cells and cause I did want to use 2 different fluorescent proteins (protein1-GFP and the other one BacMan-RFP) I proceded as follow:
1. Electroporation performed around 12 a.m., cells were seeded into a petry dish and let them adhere.
2. Around 18:00 I incubated the same cells (already adherent) with the BacMan 2.0 my protein-RFP (I used 50.000 cells to transduce with 30 PPC.) I simply added 15 uL of reagent BacMan into the DMEM, shaked a bit and let it overnight in the incubator.
3. After 17h I had a look on videomicroscope but no signal at all was detected.
Does anyone already used it? Which could be the problem? Do I have to increase the PPC up to 50?
Or...Is it bettere to add the BacMan just after eletroporation (before to seed the cell in the petrydish) ? Might the transduction be boosted?
Hoping to read your replies!
Thank you
Dear Avinash, thank you much for your reply. I’ll do that frist and if increasing the PPC does not work I wil follow the second advice. Is the confluence % a problem? Which is the best %?
Hi Daniele Campisi, I am using Thermo Fisher's Premo FUCCI to transduce epithelial cells with cell cycle proteins with GFP and RFP, and I can not get any fluorescent signal. I have already increased the PPC ratio, the incubation time, I have placed less confluence% and I do not get results. Did you manage to optimize your protocol?
Dear Raul, i could not get better results. I just increased the PPC uo to 100, less desity etc and I saw a weak signal but was not enought to get good images. So, i given up about the baculovirus!
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus