Greetings I would have thought that freezing and thawing a lump of tissue would result in all dead cells, making a suspension of fresh cells then freezing using DMSO and FCS in medium should give you much better viability after thawing. If I were you I would use an enzymic approach to making a cell suspension. collagenase and/or dispase and/or trysin would do the job, keeping your cells in divalent cation free buffer helps prevent clumping as does a tad of DNase (10u/ml). As I remember several of the collagenases in the Sigma catalog are described as suitable for making cell suspensions from liver. Doing your surface staining, fixing in formaldehyde for an hour then fixing in 70% ethanol then RNase then PI is the way I stain lymphocytes with surface markers and PI. Ammonium chloride lysis isn't that harsh and shouldn't kill your cells. High background staining is not suprising if all you cells are dead. I have no experience of liver but make suspensions of cells from gut tissue, I actually dont use enzymes any more as they changes some of the markers I was looking at, I use the Dako medimachine, enzymes result in a better yield and better viability though. Simon Monard Aaron Diamond Center NYC
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