murine lymphatic endothelial cell purification

从小鼠真皮淋巴毛细血管中分离内皮细胞

The lymphatic system plays a pivotal role in human physiology. It is considered a vital homeostatic factor, that facilitates important functions, such as the maintenance of tissue-plasma fluid balance, immune surveillance, and lipid absorption1, as well as recently-identified functions, such as the repair ability of the heart2 and regenerative capacity of bone and hematopoiesis3. Despite the significant role of the lymphatic system, several aspects of the role of the lymphatics, as well as the molecular mechanisms governing certain physiological parameters and responses remain elusive. Moreover, lymphatic vascular defects are triggering or deteriorating factors in certain pathophysiological conditions. Well-known examples are the primary and secondary lymphedemas, depending on the genetic or non-genetic origin of the disease, respectively, while the role of the lymphatic system on primary tumor growth and metastatic dissemination is pivotal, as it can act as a conduit for metastasis and modulator of immune functions1.
The lag in the study and knowledge of the lymphatic compared to the blood vasculature is mainly due to the later discovery of the lymphatic system in comparison with the blood vascular system and the delay in the identification of lymphatic-specific molecular markers. This has greatly improved in recent decades, leading to the alteration of the conventional picture of the lymphatics at steady state and in diseased conditions1. Similar to angiogenesis, lymphangiogenesis is the formation of new lymphatic vessels from pre-existing ones and plays a pivotal role in the development of a wide spectrum of diseases4,5,6. However, unlike angiogenesis, the investigation of lymphangiogenesis has been limited to mostly in vivo models focusing on developmental vessel formation and structural defects in pathological models. The isolation of the lymphatic endothelial cells is important for in vitro studies, and this can be provided by the presented protocol.
Several protocols for lymphatic endothelial isolation from mice have been developed, which require the use of fluorescence-activated cell sorting7. The advantage of the cell sorter, where available, is the higher degree of purity, contrary to bead-based isolation, with the latter providing a higher yield. The protocol utilizes the expression of lymphatic endothelial hyaluronan receptor 1 (LYVE-1), a lymphatic endothelial marker, important for cell trafficking within the lymphatic vessels4,8. Since LYVE-1 expression is limited to the lymphatic capillaries and not the collecting lymphatic vessels, the technique is suitable for the isolation of lymphatic endothelial cells specifically from the lymphatic capillaries, contrary to other lymphatic markers, such as podoplanin, which are expressed uniformly in all lymphatic endothelial cells9. For the protocol, other important lymphatic markers that were not transmembrane, such as Prox1, were excluded.
As the physiological outcome was lymphangiogenesis, which is usually initiated at the lymphatic capillaries, LYVE-1 was selected as a target due to its selectivity in these cells and because the selected antibody provided a high yield. The enzymes used were collagenase type II, which is generally known to be more effective in collagen dissociation than type I10, and dispase, a broader protease, regularly used for dermis-epidermis separation11; however, other enzymes for tissue separation have been reported12,13 and can be used. The goal of this protocol is to describe a method for dermal lymphatic endothelial cell isolation from lymphatic capillaries of adult mice with high yield using magnetic bead purification, especially in places where fluorescence-activated cell sorting is not readily available. The method is suitable for applications where the purity of lymphatic endothelial cells can be compromised for downstream applications.

作者聚焦：基于磁珠的小鼠真皮淋巴内皮细胞分离

小鼠真皮淋巴内皮细胞分离

小鼠淋巴管内皮细胞纯化

为了纯化尿液淋巴内皮细胞，在达到约80%至90%的共流度后，使用从小鼠真皮中分离的细胞，首先使用一毫升磁珠涂层溶液在磁选机中洗涤预编码的磁珠。将珠子重悬于含有 0.1% BSA 的 150 微升 DMEM 中。接下来，用PBS洗涤60毫米组织培养板中的细胞两次。将 50 微升抗体偶联珠与 3 毫升含有 0.1% BSA 的 DMEM 混合，然后将其添加到细胞中。用生物膜密封培养皿，然后将培养皿放在振荡器上，在室温下以 10 RPM 搅拌整整 10 分钟。在用 PBS 洗涤细胞一次之前丢弃培养基。快速检查细胞以验证板上是否存在 LEC 集落。向细胞中加入一毫升tripsin EDTA，并在37摄氏度下孵育三分钟，使细胞胰蛋白酶消化。检查细胞是否成功脱离细胞。用两毫升完全LEC培养基中和溶液后。将细胞溶液转移到 5 毫升无菌聚丙烯管中。使用磁性分离器，用含有 0.1% BSA 的 3 毫升 DMEM 洗涤细胞，以去除未结合的细胞。将剩余的珠子结合细胞重悬于完全 LEC 培养基中，并将它们铺板到胶原包被的 60 毫米组织培养板上。明场和荧光成像在纯化后很容易识别出 LYVE1 阳性淋巴内皮细胞，并显示几个珠子附着在上面。细胞在纯化后 24 小时表现出低汇合度，而在纯化后 7 天表现出高度汇合度。

murine-lymphatic-endothelial-cell-purification

Research

JoVE Journal

Biology

Murine Lymphatic Endothelial Cell Purification

89 次观看. 为了纯化尿液淋巴内皮细胞，在达到约80%至90%的共流度后，使用从小鼠真皮中分离的细胞，首先使用一毫升磁珠涂层溶液在磁选机中洗涤预编码的磁珠。将珠子重悬于含有 0.1% BSA 的 150 微升 DMEM 中。接下来，用PBS洗涤60毫米组织培养板中的细胞两次。将 50 微升抗体偶联珠与 3 毫升含有 0.1% BSA 的 DMEM 混合，然后将其添加到细胞中。用生物膜密封培养皿，然后将培养皿放在...

视频: 小鼠淋巴管内皮细胞纯化

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...