cell count & viability - does DMSO affect trypanblue staining after thawing (Sep/01/2004 )
after thawing of chondrocytes we counted the cells and determined their viability by trypan blue staining as well as by our automated cell counter. the problem is, that the cell counter gives a viability of about 90% but trypan blue only 50%. does someone know whether DMSO may cause any artifacts on trypan blue staining? or does someone know any other explanation for this occurance??
Trypan blue is much more accurate. Cell Counter utilizes only "cell size" as single criteria for cell counting. I believe 50% is your number for thawed culture.
I know that the criteria for cell counting with a cell counter is the cell size, but I also know that trypan blue only can enter a cell when the cell membrane is somehow defect. And a defect cell membrane causes a change in the electrical signal of the cell counter and thus a "smaller cell".
So, we think that if trypan blue can enter the cell, the cell counter should detect those cells as dead. Are we wrong?
Unfortunately, we don't know through long-term storage, frozen/thaw process which cell death mechanism (necrotic or apoptotic or both) contributes to this outcome. It's also possible that certain cell population swellen due to initial necrotic process. Generally speaking, trypan blue is still the best (except that this dye can not get into cells caused by some treatments such as beta-amyloid peptide etc....)
50% recovery sounds about right for chondrocytes in our lab, though I wash the DMSO out by diluting the cells in 10 volumes of fresh media, pellet by centrifuging, and resuspending them again in fresh media before checking viability with trypan blue.
That said, wouldn't you count more blue (dead) cells in a DMSO-based freezing solution since the DMSO increases membrane permeability allowing the TB to flow in more easily and therefore increasing the proportion of blue cells?
In my opinion JAH is absolutely right.
I agree with Jah. That's what happens when you use DMSO based freezing medium.