I infected human neural stem cells with a RFP tagged viral construct, and wanted to sort infected and undifferentiated cells. For this I use two intracellular markers, Sox2 and Nestin. When I ran my cells in FACS, I saw that the cells are nicely positive for both markers but somehow the RFP signal was not detected - although they looked RFP+ under microscope.
So I am suspecting whether the fixation/permeabilization method would affect the fluorescence signal due to pH change? I fix with 0.1% PFA for 10min, and permeabilize with 0.1% Triton X for 10min.
Any suggestions?
Which FACS instrument are you using? Does it have a 610/20 detector coupled with the blue laser?
Another thing: are you using fresh buffered PFA solution?
It was a ready made 4% PFA, which I diluted further. And I used Aria II and III.
These instruments work for RFP so they are not the problem. Is your PFA solution very old? Is it buffered and was it stored at -20oC? I prefer fresh or new solutions made in PBS pH 7.4 (this is key) and methanol free, since any denaturant will be a potential problem for the protein.
The only time I used RFP I did with live cells (no fixation nor permeabilization) because I needed them for downstream experiments. I've seen fluorescence analysis of fixed RFP proteins but they usually show some variation depending on which RFP you are using (mCherry tends to be more reliable).
You should try different times and temperatures for fixation maybe this could help. Another possibility is using an anti-RFP antibody. That would increase the signal and eliminate variations due to RFP structure loss.
Thanks for the suggestions. I decided to sort for RFP, then do antibody stainings on RFP+ cells, as I expect them to be more than 90% positive for both markers. That would be easier than trying to optimise the conditions.
Nevertheless I faced another problem: when I sorted only RFP+ cells, without any antibody staining or fixation, the cells which were alive before the sort all die after the sort (despite 100um nozzle). I sort them in PBS-EB (EDTA and 2% final BSA). I would not expect the cells to be over sensitive that they cannot take the stress and die. Would it have something to with the sorting buffer?
Ceren
1. Have you tried to lower the pressure?
2. How much EDTA are you using?
3. Are you using a viability marker to be sure your cells are not already dead? You can use 7AAD FITC and sort for 7AAD(neg) RFP(pos) cells.
4. Is your buffer pH correct? I prefer HBSS or PBS containing HEPES.
5. Are you colecting your cells in a higher BSA concentration media? The sheath fluid dilutes your final media so you need to colect cells in a more BSA concentrated media.
6. Are you pre coating your collection tubes? Droplet containing the cell is charged, it can be attracted to the charge on the plastic. This results in the droplet hitting the side of the tube wall, and the cell dying as the small volume of liquid evaporates. To prevent this, pre-coat the tube with protein/buffer to neutralize the plastic charge. Even better, make sure that your tubes are not polystyrene.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology