Isolating and purifying liver immune cells are crucial for observing the changes in intrahepatic immune responses during the development of liver diseases and exploring the potential immunological mechanisms. Therefore, the aim of this study was to provide an optimal protocol for isolating immune cells with a high yield and less damage. We compared mechanical dissection and collagenase digestion, and the results were represented by the proportion of lymphocytes, Kupffer cells and neutrophils. The apoptosis rates of liver immune cells resulted by different isolation protocols were compared by Annexin V-staining using flow cytometric analysis. Our data indicated that the enzymatic digestion in vitro was more efficient than the mechanical dissection in vitro with a suitable collagenase IV concentration of 0.01%, and the purification of liver immune cells by a one-step density gradient centrifugation in 33% Percoll had the definite advantage of a higher proportion of the target cells. We also provided evidence that enzymatic digestion in vitro method was superior to collagenase digestion in situ for liver T lymphocytes, NK cells and NKT cells isolation and purification. This protocol was also validated in human liver samples. In conclusion, we developed an optimal protocol for isolating and purifying immune cells from mouse and human liver samples in vitro by 0.01% collagenase IV and 33% Percoll density gradient centrifugation with the advantages of higher cell yields and viability. This method provides a basis for further studying liver immune cells and liver immunity with a wide range of applications.

分离和纯化肝脏免疫细胞对于观察肝脏疾病发展过程中肝内免疫反应的变化和探索潜在的免疫机制至关重要。因此，本研究的目的是提供一种最佳方案，用于分离具有高产量和较少损伤的免疫细胞。我们比较了机械解剖和胶原酶消化，结果以淋巴细胞、枯否细胞和中性粒细胞的比例表示。使用流式细胞术分析通过膜联蛋白 V 染色比较不同分离方案导致的肝免疫细胞凋亡率。我们的数据表明体外酶消化比体外机械解剖更有效合适的胶原酶 IV 浓度为 0.01%，通过一步密度梯度离心法在 33% Percoll 中纯化肝免疫细胞具有靶细胞比例较高的明显优势。我们还提供了证据表明体外酶消化法优于原位胶原酶消化法用于肝 T 淋巴细胞、NK 细胞和 NKT 细胞的分离和纯化。该协议也在人类肝脏样本中得到验证。总之，我们开发了一种最佳方案，用于在体外从小鼠和人类肝脏样本中分离和纯化免疫细胞通过0.01%胶原酶IV和33% Percoll密度梯度离心，具有更高的细胞产量和活力的优点。该方法为进一步研究肝脏免疫细胞和肝脏免疫提供了基础，具有广泛的应用前景。