Isolation of murine hepatic lymphocytes using mechanical dissection for phenotypic and functional analysis of NK1.1⁺cells

Abstract

AIM:To choose an appropriate methods for the isolation of 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 from untreated, poly (I:C)-stimulated or ConA-stimulated mice using the two methods, respectively. The cell yield per liver was evaluated by direct counting under microscope. Effects of digestive enzymes on the surface markers involved in hepatic lymphocytes were represented by relative change rate [(percentage of post-digestionpercentage of pre-digestion)/percentage of pre-digestion]. Phenotypic analyses of the subpopulations of hepatic lymphocytes and intracellular cytokines were detected by flow cytometry. The cytotoxicity of NK cells from wild C57BL/6 or poly (I:C)-stimulated C57BL/6 mice was analyzed with a 4-h 51 Cr release assay. RESULTS:NK1.1 + cell markers, NK1.1 and DX5, were significantly down-expressed after enzymatic digestion and their 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 significantly decreased. Hepatic lymphocytes isolated by the mechanical dissection comprised more innate immune cells like NK, NKT and γδ cells in normal C57BL/6 mice.

WITHDRAWN: Natural Killer Cell Activating Receptor NKG2D Is Involved in the Immunosuppressant Effect of Mycophenolate Mofetil and Infection of Hepatitis B Virus

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Protective Role of Cannabinoid Receptor 2 Activation in Galactosamine/Lipopolysaccharide-Induced Acute Liver Failure through Regulation of Macrophage Polarization and MicroRNAs

“…The liver-infiltrating cells were isolated using a modification of a protocol described earlier (Dong et al, 2004). In brief, livers were perfused with liver perfusion medium, cut into small pieces, and digested using liver digestion medium (Gibco/Life Technologies, Grand Island, NY).…”

Comparative In Vitro and In Vivo Studies of Porcine Rotavirus G9P[13] and Human Rotavirus Wa G1P[8]

“…One Gn pig without RVA inoculation was used as a negative control. Isolation of mononuclear cells (MNCs) from the ileum, spleen, liver, MLNs, and blood was conducted as previously described (45,46). RNA extraction was done on 2 ϫ 10 6 MNCs from each tissue sample by using an RNeasy minikit (Qiagen, Valencia, CA, USA) according to the manufacturer's recommendations.…”