FACS Sorting and Protein Purification - (Sep/19/2011 )
I would like to ask if someone can pinpoint me reasons for decreased cell viability and moreover, how to avoid it. I would like to do some protein extractions from my sorts, but usually I have very low levels of alive cells and would like to try any suggestions.
Also, I am working with soft tissue, and I have read that for single cell isolation people recommend trypsin for ECM digestion although somebody told me its not a good idea to add it if I am going to do protein extraction. What do you think?
Using trypsin before FACS is not a good idea. Trypsin cuts at any site following Arg and Lys in an indiscriminate fashion. So there is a strong possibility of losing cell-surface marker which result in decreased FACS efficiency.
We use non-enzymatic celll dissociation solution to dislodge cells from adherent-monolayer cell-culture.
What is your nozzle size? If you use 100um, you may have better survival (but slower sorts) than with 70um nozzle.
Also, you may want to try Collagenase/Dispase for tissue dissociation instead of Trypsin. It is much less harmful, and won't interfere as much with your protein analysis.
Thanks for answers. I did read that trypsin its just hell raised against the proteins. I will try with Collagenase/Dispase.
My nozzle size is 70um, although how changing the nozzle size would help to obtain more cells? They are already dead once you start doing the sorting, since I am staining with PI. Although if I am wrong, which could be for sure, please correct me!
It's not so much the change in nozzle size but the reduced system pressure that is used when you increase the nozzle size. Typically a 70um tip utilizes a system pressure of 50-70PSI, while a 100um tip uses a pressure of 20-30 PSI. This decrease in pressure leads to a decrease shear stress experienced by the cells and typically leads to increased viability. In addition to this, you need to make sure you are collecting your cells into a well buffered collection media (such as HBSS + 1% BSA + 50mM HEPES). Since the PBS in the system is under high pressure, it tends to get acidic. If your cells are sensitive to this decreased pH, then they will die. However, if you add HEPES to your collection buffer, you can neutralize the pH and increase viability.
Regarding the Trypsin thing. We use a product called Accutase, which is also pretty gentle on cells.