Isolation of liver infiltrating lymphocytes    
1. Hepatic T cells were isolated from fresh liver tissue collected into primary cell system.
2. In the second method, tissue was diced using sterile blades into 1-mm³ pieces in primary cell containing 1 mg/ml collagenase and incubated at 37°C for 2 h.
3. After enzymatic digestion, tissue was passed through a nylon mesh filter (100 µm) to remove cell clumps and undissociated tissue.
4. Cells were washed three times in PBS to remove collagenase, and the cell suspension was layered on a Percol density gradient (70/30%) and centrifuged for 30 min at 2000 rpm.
5. The lymphocyte band was then removed from the interface between 30% and 70% Percoll and further washed three times in PBS.
6. Greater than 95% of cells were viable by trypan blue exclusion.
7. In the second method, blocks of fresh liver tissue (2 x 2 cm) were perforated repeatedly with a 19-gauge needle (teabagging) and then injected with PBS to flush out cells from within the liver parenchyma.
8. The resultant cell suspension was filtered through fine nylon mesh to remove tissue debris and used with no further preparation for Ab staining and FACS analysis.
9. Cells obtained were compared directly with cells from the same liver isolated by the process of collagenase digestion and Percol gradient centrifugation.

Reference
Shields, P. L., C. M. Morland, M. Salmon, S. Qin, S. G. Hubscher, D. H. Adams. 1999. Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver. J. Immunol.163:6236.