Mouse lymphocyte isolation from liver - (Sep/19/2006 )

I have some trouble with lymphocyte purification from liver. I got a lot of contamination with cell debris. By the way, the method I did try are percoll or ficoll. Does anybody have the suggestion?

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Hi puliu,

Hope the following tips help (I used it successfully to isolate mononuclear cells from S. mansoni-infected mice).

1) Digest finely chopped liver (1-2 mm3 pieces) with a mix of 200 FALGPA units/ml type II Clostridium histolyticum collagenase (Sigma), 200 FALGPA units/ml bovine testis hyaluronidase (Sigma) and 50-100 Kunitz units/ml bovine pancreas DNAse I. Usually, 30-60' at 37 C on a rocker will do.

2) Vortex gently and let the debris settle for a few secs. Take the upper sup and pass it through nylon mesh.

3) Load 10 ml cell suspension on a 3-4 ml cushion made of Lympholyte M (Accurate Chemicals) at RT and centrifuge 15' at 1000 xG (no brake).

The interface should be free of debris. If not, you may wash the interface with serum-free media and repeat step 3 once again. Good luck.
Gerardo

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