Trypan Blue and fixed cells - Help settle our lab argument (Nov/17/2008 )
We are having a debate in our lab that will affect our future protocols: One person in my lab says that it is possible to fix mammalian cells to the culture dish and stain them (X-gal) for our assay (which we do routinely), then after fixation you can trypsinize them, scrape them off and count them on a hemacytometer using Trypan blue exclusion.
I disagree, because I think that fixed cells will not be able to be trypsinized to a single cell suspension, or scraped and stay intact, and that fixed cells will all be permeable to Trypan blue.
So who is right?
Why not try it?
I think you will find that trypsin will probably work, it is definitely possible to wash fixed cells off coverslips/slides so scraping will work too.
I also think that trypan blue will stain the fixed cells, especially if you have permeabilised the cells after fixation. Wouldn't the X-gal stain also show up as blue?
Trypsin detaches the adherent cells by acting on the adhesion proteins of those cells that can adhere . . . fixation of mammalian cells on tissue culture plate does not employ those adhesion proteins .. so I guess, even if trypsin detaches the adherent cells, it should be non-specific or because of some other mechanisms. U should try it and see. .but be sure to have a control to see if it is really action of trypsin.
I have counted formalin-fixed cells with trypan blue and have seen that most of the cells do not stain and some do take stain though they are all literally dead because of the fixative. I think it is because though the membrane is fixed, they are not permeable which will need a permiabliser.
If you try, let us know also and it is worth trying. But with good controls to verify your results.
I have counted formalin-fixed cells with trypan blue and have seen that most of the cells do not stain and some do take stain though they are all literally dead because of the fixative. I think it is because though the membrane is fixed, they are not permeable which will need a permiabliser.
If you try, let us know also and it is worth trying. But with good controls to verify your results.
Here's an update for anyone who cares after I did the experiment:
Well, I think I was right. It is not a reliable method. I trypsinized a dish of cells as normal and got a reference count. I then fixed two other identical dishes in 2% glutaraldehyde in PBS for 5 minutes. After 3X PBS washes, I then put 0.25% Trypsin on one and 1 mM EDTA on the other (control for any Trypsin-EDTA effect).
After 20 minutes sitting in trypsin or EDTA at 37C, not a single cell lifted off. I went ahead and scraped them off with a sterile scraper and tried to break them up by pipetting, but that was hopeless too. Just floating clumps of cells. And even though they weren't really countable, I tried, and the numbers were all over the place.
Did U try staining them with Trypan blue. That should be unreliable too with many not taking up the stain.
And we all do care 
And we all do care
I did stain all three samples with Trypan blue as well. The normally trypsinized, unfixed cells stained as normal, >95% viability and good phase contrast. Both of the fixed samples, after being manually scraped off the dish, were also stained with Trypan, and they were unusually high, over 60% blue. In addition, they were present as enormous sheets of cells when put on the hemacytometer, making counting nearly impossible.
True.
If U should work with fixed cells then fix them in suspension. I dilute to almost 1X10^6/ml to avoid clumping.