acquisition and verification of rat neutrophil extracellular traps (nets)

高效的大鼠骨髓中性粒细胞胞外陷阱分离

Neutrophils constitute a critical subset of leukocytes that play a pivotal role in the innate immune response. They are characterized by multilobed nuclei and granules containing various proteases and antimicrobial peptides1. Neutrophils primarily function through degranulation, phagocytosis, and the formation of NETs. The observation of NETs was first made by Takei et al. in 1996 during an experiment where neutrophils were stimulated with phorbol myristate acetate (PMA)2. Subsequently, the process of NET formation was coined &#34;NETosis&#34; by Brinkmann et al.3 in 2004. Their research further illuminated the crucial role of NETs in neutrophil-mediated antimicrobial responses. NETs are web-like structures composed of chromatin, histones, and antimicrobial proteins that are released from activated neutrophils in response to infectious and inflammatory stimuli. NETs can immobilize and kill invading pathogens by trapping them and exposing them to a high concentration of antimicrobial peptides and proteases1,3. Additionally, NETs contribute to the clearance of apoptotic cells and participate in inflammation resolution. Recent studies also indicate that an excessive formation of NETs or impaired NET degradation can lead to tissue damage, autoimmune disorders, thrombogenesis, and impaired revascularization4,5,6,7,8,9,10.
The pathogenic role of NETs in uncontrolled fibrosis following myocardial infarction and the formation of ventricular aneurysms has been demonstrated through the expansion of perivascular fibrosis4,11. The myocardial infarction model and the isolation of neutrophils from bone marrow in mice are both well-established. Polymorphonuclear (PMN) leukocytes, a type of white blood cell abundant in human blood, serve as an excellent source for isolating human neutrophils. This method eliminates the need to harvest bone marrow, thus enhancing safety and efficiency.
NETs also play a role in atrial fibrillation associated with cardiac remodeling. However, large animals such as dogs and pigs were utilized to model atrial fibrillation, as mice lack an atrium sizable enough to establish a re-entrant cycle or the AF model, unless specific ion channels or signaling pathways are knocked down or knocked out12. While it&#39;s possible to induce atrial fibrillation in rats and isolate neutrophils from rat peripheral blood as previously described, researchers encountered a limitation whereby only 2 x 105-5 x 105 neutrophils could be isolated from peripheral blood (10 mL per rat). Extracting sufficient NETs at each time point required approximately 10-25 rats (5 x 106 neutrophils in total), resulting in a time-consuming, expensive, and often low-yield process13. In this regard, Li He and colleagues present a bone marrow-oriented strategy to obtain adequate NETs from rats14. In their article, they provide a comprehensive description of isolating neutrophils from rat bone marrow and compare the NET secretion capabilities of rat peripheral and bone marrow neutrophils. The two methods outlined cater to distinct experimental goals, both resulting in sufficient quantities of rat bone marrow neutrophils while reducing the number of required rats. The two-step isolation method demonstrated superior neutrophil purification, while the one-step method proved time-efficient with acceptable purification levels. Furthermore, the researchers compared NETosis and NET formation between rat bone marrow neutrophils and their peripheral counterparts, finding equal potency with PMN. These findings significantly contribute to neutrophil-related studies of atrial fibrillation and underscore the importance of flexibly selecting different sources for neutrophil isolation in various experimental animals with differing neutrophil distributions.

作者聚焦：从骨髓中分离大鼠中性粒细胞和 NET 的优化方法

分离具有相当容量的大鼠骨髓中性粒细胞的独特方法

Watch this Scientific Journal Video about Acquisition and Verification of Rat Neutrophil Extracellular Traps NETs at JoVE.com

大鼠中性粒细胞胞外诱捕器（NETs）的采集和验证

首先，将分离的大鼠中性粒细胞重悬于无菌10厘米培养皿中的四毫升补充RPMI培养基中。接下来，向中性粒细胞悬浮液中加入四微升PMA以触发NET的形成。将混合物在 37% 二氧化碳下在 5% 的二氧化碳下孵育 3 小时。对于阴性对照，在中性粒细胞悬浮液中加入 4 至 5 微升 DNASE1 以降解分泌的 NET。然后，加入四毫升HBSS洗涤NET。用每块板的 4 毫升新鲜培养基冲洗板以分离 NET。现在，将洗涤介质收集在新管中并经常移液，以确保NET完全重新悬浮。将悬浮液在300g下在20摄氏度下离心10分钟，以除去任何漂浮的细胞。然后，将含有NET的上清液培养基收集在新管中。将NET和浮动细胞的悬浮液离心在24孔板中，并在其中放置1.4厘米的玻璃盖玻片以沉降任何浮动细胞。然后，用 4% PFA 将细胞固定在盖玻片上，并继续验证 NET 的存在。接下来，将一滴HBSS放在石蜡膜覆盖的试管架上。将盖玻片倒入 HBSS 液滴中进行清洗。洗涤后，将细胞在室温下在250微升0.5x Triton X-100中孵育10分钟以使其透化。然后，在HBSS中洗涤细胞三次，每次一分钟。将细胞在室温下密封在10%正常驴血清中一小时。最后，将细胞与 70 至 90 微升抗大鼠抗体在 4 摄氏度下孵育过夜。在 HBSS 中清洗盖玻片 3 次，每次 5 分钟。现在，在洗涤HBSS之前，在室温下将盖玻片在二抗中在黑暗中孵育一小时。将细胞核和 NET 骨架染色在 70 至 90 微升 DAPI 中。进行 HBSS 洗涤 3 次，每次 5 分钟。

acquisition-verification-rat-neutrophil-extracellular-traps

Research

JoVE Journal

Biology

Acquisition and Verification of Rat Neutrophil Extracellular Traps (NETs)

108 次观看. 首先，将分离的大鼠中性粒细胞重悬于无菌10厘米培养皿中的四毫升补充RPMI培养基中。接下来，向中性粒细胞悬浮液中加入四微升PMA以触发NET的形成。将混合物在 37% 二氧化碳下在 5% 的二氧化碳下孵育 3 小时。对于阴性对照，在中性粒细胞悬浮液中加入 4 至 5 微升 DNASE1 以降解分泌的 NET。然后，加入四毫升HBSS洗涤NET。用每块板的 4 毫升新鲜培养基冲洗板以分离 NET。

视频: 大鼠中性粒细胞胞外诱捕器（NETs）的采集和验证

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone marrow. This effort arose due to limitations associated with the traditional method of extracting NETs from peripheral blood, mainly due to the scarcity of available neutrophils for isolation. The study revealed two distinct methodologies for obtaining rat neutrophils from bone marrow: a streamlined one-step procedure that yielded satisfactory purification levels, and a more time-intensive two-step process that exhibited enhanced purification efficiency. Importantly, both techniques yielded a substantial quantity of viable neutrophils, ranging between 50 to 100 million per rat. This efficiency mirrored the results obtained from isolating neutrophils from both human and murine sources. Significantly, neutrophils derived from rat bone marrow exhibited comparable abilities to secrete NETs when compared with neutrophils obtained from peripheral blood. However, the bone marrow-based method consistently produced notably larger quantities of both neutrophils and NETs. This approach demonstrated the potential to obtain significantly greater amounts of these cellular components for further downstream applications. Notably, these isolated NETs and neutrophils hold promise for a range of applications, spanning the realms of inflammation, infection, and autoimmune diseases.

This research outlines two techniques for isolating abundant neutrophil extracellular traps (NETs) from rat bone marrow. One method combines a commercial neutrophil isolation kit with density gradient centrifugation, while the other employs only density gradient centrifugation. Both approaches yield functional NETs surpassing those from peripheral blood neutrophils.

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone ...