Name: 好中球細胞外トラップ(NET)を採取するためのラット好中球単離の1ステップおよび2ステップ法
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)

効率的なラット骨髄好中球細胞外トラップの単離

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.

著者スポットライト:骨髄からラット好中球およびNETを分離するための最適化された方法(英語)

同等の容量を持つラット骨髄好中球を単離するためのユニークなアプローチ

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

好中球細胞外トラップ(NET)を採取するためのラット好中球単離の1ステップおよび2ステップ法

骨髄採取を開始するには、安楽死させたラットを75%エタノールに浸し、毛皮と皮膚を殺菌します。ネズミを仰向けに寝かせ、腰で下肢を切断して大腿骨の頭を保護します。解剖ハサミを使用して、ホックジョイントの靭帯を切断します。次に、膝関節を後方に折りたたんで、大腿骨と脛骨を露出させます。鉗子とハサミを使用して、骨に接続されている筋肉、腱、その他の組織を慎重に取り除きます。5ミリメートルの針状注射器で骨の両端に骨髄を突き刺し、骨髄マトリックスを緩めます。次に、新しいシリンジを使用して、10〜15ミリリットルのRPMI培地で骨髄を洗い流します。骨髄細胞を300Gで20°Cで10分間遠心分離します。上清を廃棄し、細胞を5〜10ミリリットルの赤血球溶解緩衝液に再懸濁します。懸濁液をインキュベートした後、混合物に4倍の容量のHBSSを加えます。その後、細胞を600Gで室温で5分間遠心分離します。得られたペレットをさらに使用してください。まず、ラット骨髄好中球分離キットで好中球を単離します。15ミリリットルの遠心分離管に80〜55%の80-55%の試薬を2ミリリットル重ねて、密度勾配を作成します。次に、単離された好中球、またはラット骨髄好中球分離キットで単離された細胞の再懸濁液をチューブにピペットで移します。チューブを800Gで20°Cで40分間遠心分離します。滅菌ピペットを使用して、65〜70%境界の細胞層を含む70%グラジエント層を回収します。細胞懸濁液を新しい15ミリリットルの遠心チューブに移します。15ミリリットルのHBSSをチューブに加え、静かに反転させて内容物を混ぜます。最後に、チューブを室温で300Gで10分間遠心分離し、細胞をペレット化します。ワンステッププロセスでは、骨髄懸濁液を密度勾配チューブに直接ピペッティングした後、前述のように遠心分離します。次に、75〜70%のグラデーション境界にあるレイヤーを含む70%グラデーションレイヤーをピペットで取り出します。この細胞懸濁液を、10ミリリットルのHBSSを入れた新しい50ミリリットルの遠心チューブに移します。次に、チューブを400Gで室温で5分間遠心分離し、細胞をペレット化します。単離された好中球を血球計算盤でカウントし、トリパンブルー染色で細胞の生存率を評価します。2 ステップ法では、1 ステップ法で達成された 50% と比較して、90% の純度レベルが向上しました。

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 ...