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.