Name: Acquisition et vérification de pièges extracellulaires à neutrophiles (TNE) chez le rat
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)

Isolation efficace du piège extracellulaire de neutrophiles de moelle osseuse de rat

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.

Pleins feux sur l’auteur : Méthode optimisée pour isoler les neutrophiles et les TNE de rat de la moelle osseuse

Approche unique pour isoler les neutrophiles de la moelle osseuse du rat avec une capacité comparable

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

Acquisition et vérification de pièges extracellulaires à neutrophiles (TNE) chez le rat

Pour commencer, mettez en suspension les neutrophiles de rat isolés dans quatre millilitres de milieu RPMI supplémenté dans une boîte de Pétri stérile de 10 centimètres. Ensuite, ajoutez quatre microlitres de PMA à la suspension de neutrophiles pour déclencher la formation de TNE. Incuber le mélange à 37 degrés Celsius pendant trois heures sous 5% de dioxyde de carbone.Pour le contrôle négatif, ajoutez quatre à cinq microlitres d’ADNASE1 à la suspension de neutrophiles pour dégrader les TNE sécrétées. Ensuite, ajoutez quatre millilitres de HBSS pour laver les TNE. Rincer la plaque avec quatre millilitres de milieu de culture frais pour chaque plaque afin de détacher les TNE.Maintenant, récupérez le produit de lavage dans un nouveau tube et pipetez fréquemment pour assurer une remise en suspension complète des TNE. Centrifugez la suspension à 300 g pendant 10 minutes à 20 degrés Celsius pour éliminer les cellules flottantes. Ensuite, prélevez le milieu surnageant avec les TNE dans un nouveau tube.Centrifuger la suspension des TNE et des cellules flottantes dans une plaque de 24 puits avec des lamelles de verre de 1,4 centimètre placées pour déposer les cellules flottantes. Ensuite, fixez les cellules sur les lamelles avec 4% de PFA et procédez à la vérification de la présence des TNE. Ensuite, placez une goutte de HBSS sur un support de tube à essai recouvert d’un film de paraffine.Retournez la housse dans les gouttelettes HBSS pour la laver. Après le lavage, incubez les cellules dans 250 microlitres de 0,5x Triton X-100 pendant 10 minutes à température ambiante pour les perméabiliser. Ensuite, lavez les cellules trois fois dans HBSS pendant une minute.Scellez les cellules dans du sérum d’ânesse normal à 10% à température ambiante pendant une heure. Enfin, incubez les cellules avec 70 à 90 microlitres d’anticorps anti-rat pendant la nuit à quatre degrés Celsius. Lavez la lamelle dans HBSS trois fois pendant cinq minutes chacune.Maintenant, incubez la lamelle dans les anticorps secondaires à température ambiante pendant une heure dans l’obscurité avant de laver un HBSS. Colorer les noyaux cellulaires et les squelettes des TNE dans 70 à 90 microlitres de DAPI. Effectuez le lavage HBSS trois fois pendant cinq minutes chacune.

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 ...