Name: Acquisizione e verifica di trappole extracellulari per neutrofili di ratto (NET)
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)

Isolamento efficiente della trappola extracellulare dei neutrofili del midollo osseo di ratto

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.

Autore in primo piano: Metodo ottimizzato per l'isolamento di neutrofili e NET di ratto dal midollo osseo

Approccio unico per l'isolamento di neutrofili del midollo osseo di ratto con capacità comparabili

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

Acquisizione e verifica di trappole extracellulari per neutrofili di ratto (NET)

Per iniziare, risospendere i neutrofili di ratto isolati in quattro millilitri di terreno RPMI integrato in una capsula di Petri sterile da 10 centimetri. Successivamente, aggiungere quattro microlitri di PMA alla sospensione dei neutrofili per innescare la formazione di NET. Incubare la miscela a 37 gradi Celsius per tre ore sotto il 5% di anidride carbonica.Per il controllo negativo, aggiungere da quattro a cinque microlitri di DNASE1 alla sospensione dei neutrofili per degradare i NET secreti. Quindi, aggiungi quattro millilitri di HBSS per lavare i NET. Sciacquare la piastra con quattro millilitri di terreno di coltura fresco per ciascuna piastra per staccare i NET.A questo punto, raccogliere frequentemente il mezzo di lavaggio in una nuova provetta e pipetta per garantire la completa risospensione dei NET. Centrifugare la sospensione a 300 g per 10 minuti a 20 gradi Celsius per rimuovere eventuali cellule galleggianti. Quindi, raccogliere il terreno surnatante con i NET in una nuova provetta.Centrifugare la sospensione di NET e cellule galleggianti in una piastra da 24 pozzetti con vetrini di vetro da 1,4 centimetri posizionati per depositare eventuali cellule galleggianti. Quindi, fissare le celle sui vetrini coprioggetto con PFA al 4% e procedere alla verifica della presenza dei NET. Successivamente, posizionare una goccia di HBSS su un supporto per provette ricoperto di film di paraffina.Capovolgere la vetrina di copertura nelle goccioline HBSS per lavarla. Dopo il lavaggio, incubare le cellule in 250 microlitri di Triton X-100 0,5x per 10 minuti a temperatura ambiente per permeabilizzarle. Quindi, lavare le cellule tre volte in HBSS per un minuto.Sigillare le cellule in siero d'asina normale al 10% a temperatura ambiente per un'ora. Infine, incubare le cellule con 70-90 microlitri di anticorpi anti-ratto durante la notte a quattro gradi Celsius. Lavare il vetrino coprioggetto in HBSS tre volte per cinque minuti ciascuna.Ora, incubare il vetrino coprioggetto negli anticorpi secondari a temperatura ambiente per un'ora al buio prima di lavare un HBSS. Colorare i nuclei cellulari e gli scheletri di NET in 70-90 microlitri di DAPI. Eseguire il lavaggio HBSS tre volte per cinque minuti ciascuna.

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 ...