Name: 호중구 세포외 트랩(NET) 채취를 위한 쥐 호중구 분리의 1단계 및 2단계 방법
Uploaded: 2024-04-26T12:30:00
Description: Watch this Scientific Journal Video about One and Two-Step Method of Rat Neutrophil Isolation for Harvesting Neutrophil Extracellular Traps NETs at JoVE.com

one and two-step method of rat neutrophil isolation for harvesting neutrophil extracellular traps (nets)

효율적인 Rat Bone Marrow Neutrophil Extracellular Trap 분리

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.

저자 스포트라이트: 골수에서 쥐 호중구와 NET을 분리하기 위한 최적화된 방법

비슷한 용량의 쥐 골수 호중구를 분리하기 위한 독특한 접근법

We have developed a method of isolating rat neutrophils and subsequently extracting NETs. This method offers to optimize the accessibility for conducting NETs-related experiments in both rats and rat-derived cell lines. In our study, we conducted a comparation between neutrophils that were isolated from the peripheral blood and bone marrow threads.Our findings highlight the superiority of bone marrow-derived neutrophils, showcasing a substantial numerical advantage in isolated neutrophil counts alongside their proficient capability to synchronize. Our bone marrow-based method for isolating rat neutrophils and NETs fills a research gap by optimizing isolation techniques for these cell types. Unlike previous methods that primarily used peripheral blood, our protocol targets bone marrow, increasing neutrophil in large yields, and offering a more accurate rat model for studying neutrophil and rat biology.

Watch this Scientific Journal Video about One and Two-Step Method of Rat Neutrophil Isolation for Harvesting Neutrophil Extracellular Traps NETs at JoVE.com

호중구 세포외 트랩(NET) 채취를 위한 쥐 호중구 분리의 1단계 및 2단계 방법

골수 채취를 시작하려면 안락사된 쥐를 75% 에탄올에 담가 털과 피부를 소독합니다. 쥐를 눕히고 엉덩이에서 하지를 절단하여 대퇴골 머리를 보호합니다. 해부 가위를 사용하여 호크 관절의 인대를 절단하십시오.그런 다음 무릎 관절을 뒤로 접어 대퇴골과 경골을 노출시킵니다. 겸자와 가위를 사용하여 뼈에 연결된 근육, 힘줄 및 기타 조직을 조심스럽게 제거합니다. 5mm 바늘 주사기로 양쪽 뼈 끝을 통해 골수를 찔러 골수 매트릭스를 풉니다.이제 새 주사기를 사용하여 10-15ml의 RPMI 배지로 골수를 씻어냅니다. 골수 세포를 섭씨 300도에서 10분 동안 20G로 원심분리합니다. 상층액을 버리고 5-10 밀리리터의 적혈구 용해 완충액에 세포를 재현탁시킵니다.현탁액을 배양한 후 혼합물에 HBSS 부피의 4배를 추가합니다. 그런 다음 실온에서 5분 동안 600G에서 세포를 원심분리합니다. 생성된 펠릿을 추가 사용을 위해 사용하십시오.먼저 쥐 골수 호중구 분리 키트로 호중구를 분리합니다. 밀도 구배를 만들기 위해 15 밀리리터 원심 분리 튜브에 col 시약 당 80-55 %의 2 밀리리터를 층층화합니다. 그런 다음 분리된 호중구 또는 쥐 골수 호중구 분리 키트로 분리된 세포의 재현탁액을 튜브에 피펫팅합니다.튜브를 섭씨 800도에서 40분 동안 20G로 원심분리합니다. 멸균 피펫을 사용하여 65-70% 경계에 있는 세포층을 포함하여 70% 그래디언트 층을 수집합니다. 셀 현탁액을 새 15밀리리터 원심분리기 튜브로 옮깁니다.튜브에 HBSS 15mL를 넣고 부드럽게 뒤집어 내용물을 섞습니다. 마지막으로 튜브를 실온에서 300G에서 10분 동안 원심분리하여 세포를 펠렛화합니다. 원스텝 공정의 경우, 골수 현탁액을 밀도 구배 튜브에 직접 피펫팅한 후 앞서 설명한 대로 원심분리합니다.다음으로, 75-70% 그래디언트 경계에 있는 레이어를 포함하여 70% 그래디언트 레이어를 피펫팅합니다. 이 셀 현탁액을 10ml의 HBSS가 들어 있는 새로운 50mL 원심분리기 튜브로 옮깁니다. 그런 다음 튜브를 실온에서 400G에서 5분 동안 원심분리하여 세포를 펠릿화합니다.혈구계를 사용하여 분리된 호중구를 계수하고 트리판 블루 염색을 통해 세포 생존율을 평가합니다. 2단계 방법은 1단계 방법에 의해 달성된 50%에 비해 90%as의 향상된 순도 수준을 가졌습니다.

Research

JoVE Journal

Biology

One and Two-Step Method of Rat Neutrophil Isolation for Harvesting Neutrophil Extracellular Traps (NETs)

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 ...