Name: Et- og totrinsmetode til rotteneutrofilisolering til høst af neutrofile ekstracellulære fælder (NET'er)
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)

Effektiv rotteknoglemarv Neutrofil ekstracellulær fældeisolering

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.

Forfatter Spotlight: Optimeret metode til isolering af rotteneutrofiler og NET'er fra knoglemarv

Unik tilgang til isolering af neutrofiler i rotteknoglemarv med sammenlignelig kapacitet

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

Et- og totrinsmetode til rotteneutrofilisolering til høst af neutrofile ekstracellulære fælder (NET'er)

For at begynde knoglemarvshøsten skal du lægge en aflivet rotte i blød i 75% ethanol for at sterilisere pels og hud. Læg rotten på ryggen og afskær underbenene ved hoften for at beskytte lårbenshovedet. Brug dissektionssaks til at skære gennem ledbåndet ved haseleddet.Fold derefter knæleddet bagud for at udsætte lårbenet og skinnebenet. Med et par tang og saks skal du forsigtigt fjerne muskler, sener og andet væv, der er forbundet med knoglerne. Stik marven gennem begge knogleender med en 5 millimeter nålet sprøjte for at løsne marvmatrixen.Brug nu en frisk sprøjte til at skylle knoglemarven ud med 10-15 ml RPMI-medier. Centrifuger knoglemarvscellerne ved 300 G i 10 minutter ved 20 grader Celsius. Supernatanten kasseres, og cellerne resuspenderes i 5-10 ml lysebuffer for røde blodlegemer.Efter inkubation af suspensionen tilsættes fire gange volumenet HBSS til blandingen. Derefter centrifugeres cellerne ved 600G i fem minutter ved stuetemperatur. Brug den resulterende pellet til videre brug.Isoler neutrofilerne først med neutrofilisoleringssæt til rotteknoglemarv. Lag 2 ml 80-55% pr. Col reagens i et 15 ml centrifugerør for at skabe en densitetsgradient. Derefter pipetteres de isolerede neutrofiler eller resuspensionsopløsningen af celler isoleret med rotteknoglemarvsneutrofilisoleringssæt ind i røret.Centrifuger røret ved 800G i 40 minutter ved 20 grader Celsius. Med en steril pipette opsamles 70%gradientlaget, inklusive cellelagene ved 65-70%-grænsen. Overfør cellesuspensionen til et nyt 15 ml centrifugerør.Tilsæt 15 ml HBSS i røret og vend forsigtigt for at blande indholdet. Til sidst centrifugeres røret ved 300 G i 10 minutter ved stuetemperatur for at pelletere cellerne. For ettrinsprocessen centrifugeres knoglemarvssuspensionen direkte ind i densitetsgradientrøret efter pipettering som tidligere vist.Derefter pipetteres 70%gradientlaget, inklusive lagene ved 75-70%gradientgrænsen. Overfør denne cellesuspension til et nyt 50 ml centrifugerør med 10 ml HBSS. Derefter centrifugeres røret ved 400G i fem minutter ved stuetemperatur for at pelletere cellerne.Tæl de isolerede neutrofiler ved hjælp af et hæmocytometer, og vurder cellelevedygtigheden via trypanblå farvning. Totrinsmetoden havde et forbedret renhedsniveau på 90% i forhold til de 50%, der blev opnået ved ettrinsmetoden.

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 ...