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AIM:To choose an appropriate methods for the isolationof hepatic lymphocytes between the mechanical dissectionand the enzymatic digestion and investigate the effects oftwo methods on phenotype and function of hepaticlymphocytes.METHODS:Hepatic lymphocytes were isolated fromuntreated,poly (I:C)-stimulated or ConA-stimulated miceusing the two methods,respectively.The cell yield perliver was evaluated by direct counting under microscope.Effects of digestive enzymes on the surface markersinvolved in hepatic lymphocytes were represented byrelative change rate [(percentage of post-digestion-percentage of pre-digestion)/percentage of pre-digestion].Phenotypic analyses of the subpopulations of hepaticlymphocytes and intracellular cytokines were detected byflow cytometry.The cytotoxicity of NK cells from wildC57BL/6 or poly (I:C)-stimulated C57BL/6 mice wasanalyzed with a 4-h ~(51)Cr release assay.RESULTS:NK1.1~+ cell markers,NK1.1 and DX5,weresignificantly down-expressed after enzymatic digestion andtheir relative change rates were about 28% and 32%,respectively.Compared with the enzymatic digestion,thecell yield isolated from unstimulated,poly (I:C)-treated orConA-treated mice by mechanical dissection was notsignificantly decreased.Hepatic lymphocytes isolated bythe mechanical dissection comprised more innate immunecells like NK,NKT and γδ cells in normal C57BL/6 mice.After poly (I:C) stimulation,hepatic NK cells rose to about35%,while NKT cells simultaneously decreased.FollowingConA injection,the number of hepatic NKT cells wasremarkably reduced to 3.67%.Higher ratio of intracellularIFN-γ~+(68%) or TNF-α~+(15%) NK1.1~+ cells from poly (I:C)-treated mice was obtained using mechanical dissectionmethod than control mice.There was no difference in viability between the mechanical dissection and theenzymatic digestion,and hepatic lymphocytes obtained withthe two methods had similar cytotoxicity against YAC-1cells.CONCLUSION:There is no difference in the cell yield andviability of the hepatic lymphocyte isolated with the twomethods.The mechanical dissection,but not the enzymaticdigestion,may be suitable for the phenotypic analysis ofhepatic NK1.1~+ cell.
AIM: To choose an appropriate method for the isolationof hepatic lymphocytes between the mechanical dissection and the enzymatic digestion and investigate the effects of two methods on phenotype and function of hepatic lymphocytes. METHODS: Hepatic lymphocytes were isolated fromuntreated, poly (I: C) -stimulated or ConA -stimulated miceusing the two methods, respectively. cell yield perliver was evaluated by direct counting under microscope. Effects of digestive enzymes on the surface markers in viral in hepatic lymphocytes were represented by relative change rate [(percentage of post-digestion-percentage of pre-digestion ) / percentage of pre-digestion]. Phenotypic analyzes of the subpopulations of hepaticlymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wildC57BL / 6 or poly (I: C) -stimulated C57BL / 6 mice was wasalyzed with a 4 -h ~ (51) Cr release assay .RESULTS: NK1.1 ~ + cell markers, NK1.1 and DX5, weresignificantly down-expressed after enzymatic digest ion and the relative change rates were about 28% and 32%, respectively. Compared with the enzymatic digestion, the cell yield isolated from unstimulated, poly (I: C) -treated or CONA-treated mice by mechanical dissection was not statistically reduced compared tothethe mechanical dissection was more innate immune cells like NK, NKT and γδ cells in normal C57BL / 6 mice. After poly (I: C) stimulation, hepatic NK cells rose to about 35% NKT cells was significantly reduced to 3.67% .Higher ratio of intracellular IFN- [gamma] + (68%) or TNF- [alpha] + (15%) NK1.1 ~ + cells from poly dissectionmethod than control mice .here was no difference in viability between the mechanical dissection and theenzymatic digestion, and hepatic lymphocytes obtained with the two methods were similar cytotoxicity against YAC-1 cells. CONCLUSION: There is no difference in the cell yield andviabi lity of the hepatic lymphocyte isolated with the twomethods.The mechanical dissection, but not the enzymatic digestion, may be suitable for the phenotypic analysis of hepatic NK1.1 ~ + cell.