Name: Erfassung und Verifizierung von extrazellulären Fallen (NETs) neutrophiler Ratten
Uploaded: 2024-04-26T12:30:00
Description: Watch this Scientific Journal Video about Acquisition and Verification of Rat Neutrophil Extracellular Traps NETs at JoVE.com

acquisition and verification of rat neutrophil extracellular traps (nets)

Effiziente Isolierung von extrazellulären Fallen aus dem Knochenmark von Ratten mit Neutrophilen

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.

Autor im Rampenlicht: Optimierte Methode zur Isolierung von Neutrophilen und NETs von Ratten aus dem Knochenmark

Einzigartiger Ansatz zur Isolierung von Neutrophilen im Knochenmark von Ratten mit vergleichbarer Kapazität

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 Acquisition and Verification of Rat Neutrophil Extracellular Traps NETs at JoVE.com

Erfassung und Verifizierung von extrazellulären Fallen (NETs) neutrophiler Ratten

Zu Beginn resuspendieren Sie die isolierten Rattenneutrophilen in vier Millilitern supplementierter RPMI-Medien in einer sterilen 10-Zentimeter-Petrischale. Als nächstes fügen Sie der neutrophilen Suspension vier Mikroliter PMA hinzu, um die Bildung von NETs auszulösen. Inkubieren Sie die Mischung drei Stunden lang bei 37 Grad Celsius unter 5% Kohlendioxid.Für die Negativkontrolle fügen Sie der Neutrophilensuspension vier bis fünf Mikroliter DNASE1 hinzu, um die sezernierten NETs abzubauen. Fügen Sie dann vier Milliliter HBSS hinzu, um die NETs zu waschen. Spülen Sie die Platte mit vier Millilitern frischem Nährmedium für jede Platte, um die NETs zu lösen.Sammeln Sie nun das Waschmedium in einem neuen Röhrchen und pipettieren Sie es häufig, um eine vollständige Resuspension der NETs zu gewährleisten. Die Suspension bei 300 g 10 Minuten bei 20 Grad Celsius zentrifugieren, um alle schwimmenden Zellen zu entfernen. Sammeln Sie dann das überstehende Medium mit NETs in einem neuen Rohr.Zentrifugieren Sie die Suspension von NETs und schwimmenden Zellen in einer 24-Well-Platte mit 1,4 Zentimeter großen Glasdeckgläsern, um schwimmende Zellen abzusetzen. Befestigen Sie dann die Zellen auf Deckgläsern mit 4 % PFA und überprüfen Sie das Vorhandensein der NETs. Geben Sie als Nächstes einen Tropfen HBSS auf einen mit Paraffinfilm bedeckten Reagenzglasständer.Drehen Sie den Eindeckbezug in die HBSS-Tröpfchen, um ihn zu waschen. Nach dem Waschen die Zellen in 250 Mikrolitern 0,5x Triton X-100 10 Minuten bei Raumtemperatur inkubieren, um sie zu permeabilisieren. Waschen Sie dann die Zellen dreimal eine Minute lang in HBSS.Verschließen Sie die Zellen in 10% normalem Eselsserum bei Raumtemperatur für eine Stunde. Schließlich inkubieren Sie die Zellen mit 70 bis 90 Mikrolitern Anti-Ratten-Antikörpern über Nacht bei vier Grad Celsius. Waschen Sie den Deckbezug dreimal für jeweils fünf Minuten in HBSS.Inkubieren Sie nun das Deckglas in den sekundären Antikörpern bei Raumtemperatur eine Stunde lang im Dunkeln, bevor Sie ein HBSS waschen. Färben Sie die Zellkerne und NETs-Skelette in 70 bis 90 Mikrolitern DAPI. Führen Sie die HBSS-Wäsche dreimal für jeweils fünf Minuten durch.

Research

JoVE Journal

Biology

Acquisition and Verification of Rat 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 ...