Name: Metodo in una e due fasi di isolamento dei neutrofili di ratto per la raccolta di trappole extracellulari di neutrofili (NET)
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)

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 One and Two-Step Method of Rat Neutrophil Isolation for Harvesting Neutrophil Extracellular Traps NETs at JoVE.com

Metodo in una e due fasi di isolamento dei neutrofili di ratto per la raccolta di trappole extracellulari di neutrofili (NET)

Per iniziare il prelievo del midollo osseo, immergere un ratto soppresso in etanolo al 75% per sterilizzare il pelo e la pelle. Appoggia il ratto sulla schiena e recisci gli arti inferiori all'altezza dell'anca per proteggere la testa del femore. Usa le forbici da dissezione per tagliare il legamento all'articolazione del garretto.Quindi piegare l'articolazione del ginocchio all'indietro per esporre il femore e la tibia. Con un paio di pinze e forbici, rimuovi con cura eventuali muscoli, tendini e altri tessuti collegati alle ossa. Infilare il midollo attraverso entrambe le estremità ossee con una siringa agugliata da 5 millimetri per allentare la matrice del midollo.Ora usa una siringa nuova per risciacquare il midollo osseo con 10-15 millilitri di terreno RPMI. Centrifugare le cellule del midollo osseo a 300 G per 10 minuti a 20 gradi Celsius. Scartare il surnatante e risospendere le cellule in 5-10 millilitri di tampone di lisi dei globuli rossi.Dopo aver incubato la sospensione, aggiungere quattro volte il volume di HBSS alla miscela. Quindi centrifugare le celle a 600 G per cinque minuti a temperatura ambiente. Utilizzare il pellet risultante per un ulteriore utilizzo.Isolare prima i neutrofili con il kit di isolamento dei neutrofili del midollo osseo di ratto. Stratificare 2 millilitri di reagente all'80-55% per col in una provetta da centrifuga da 15 millilitri, per creare un gradiente di densità. Quindi pipettare i neutrofili isolati, o la soluzione di risospensione di cellule isolate con il kit di isolamento dei neutrofili del midollo osseo di ratto, nella provetta.Centrifugare la provetta a 800 G per 40 minuti a 20 gradi Celsius. Con una pipetta sterile, raccogliere lo strato di gradiente del 70%, compresi gli strati cellulari al limite del 65-70%. Trasferire la sospensione cellulare in una nuova provetta da centrifuga da 15 millilitri.Aggiungere 15 millilitri di HBSS nel tubo e capovolgere delicatamente per mescolarne il contenuto. Infine, centrifugare la provetta a 300G per 10 minuti a temperatura ambiente per pellettare le cellule. Per il processo in un'unica fase, dopo aver pipettato la sospensione del midollo osseo direttamente nella provetta a gradiente di densità, centrifugare come dimostrato in precedenza.Quindi, pipettare lo strato di gradiente del 70%, compresi gli strati al limite del gradiente del 75-70%. Trasferire questa sospensione cellulare in una nuova provetta da centrifuga da 50 millilitri con 10 millilitri di HBSS. Quindi centrifugare la provetta a 400 G per cinque minuti a temperatura ambiente per pellettizzare le cellule.Contare i neutrofili isolati utilizzando un emocitometro e valutare la vitalità cellulare tramite la colorazione blu tripano. Il metodo in due fasi ha avuto un livello di purezza superiore del 90% rispetto al 50% ottenuto dal metodo in una fase.

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 ...