facs enrichment of zebrafish gut cell suspension for scrnaseq

Elaborazione di cellule intestinali da larve di pesce zebra per il sequenziamento dell'RNA a cellula singola

The gastrointestinal (GI) tract is a complex system that plays a vital role in overall health and well-being. It is responsible for the digestion and absorption of nutrients, as well as the elimination of waste products1,2. The GI tract is composed of multiple cell types, including epithelial cells, smooth muscle cells, immune cells, and the enteric nervous system (ENS), which communicate closely together to regulate and maintain proper intestinal function3,4,5. Defects in the development of the GI tract can have far-reaching effects on various aspects such as nutrient absorption, microbiota composition, the gut-brain axis, and the ENS, leading to several enteric neuromuscular disorders, such as Hirschsprung disease and Chronic intestinal pseudo-obstruction6,7. These disorders are characterized by severe gut dysmotility caused by alterations in various key cells, such as the interstitial cells of Cajal, smooth muscle cells, and the ENS6,8,9. However, the molecular mechanisms underlying GI development and dysfunction are still poorly understood.
The zebrafish is a valuable model organism for studying GI development and dysfunction due to its rapid embryonic development, transparency during embryonic and larval stages, and genetic tractability10,11,12,13,14. Numerous transgenic zebrafish lines expressing fluorescent proteins are available. An example of such a line is the tg(phox2bb:GFP) zebrafish, commonly used to study the ENS, as all phox2bb+ cells, including enteric neurons, are labeled15,16. Here, using the tg(phox2bb:GFP) zebrafish line, we present a method for intestinal isolation of 5 days post fertilization (dpf) larvae for single-cell RNA sequencing (scRNA-seq) analysis (Figure 1).

Autore in primo piano: Isolamento e analisi delle cellule intestinali delle larve di zebrafish per lo studio degli aspetti trascrittomici dello sviluppo gastrointestinale 

Isolamento intestinale da larve di pesce zebra per il sequenziamento dell'RNA a singola cellula

Watch this Scientific Journal Video about FACS Enrichment of Zebrafish Gut Cell Suspension for scRNAseq at JoVE.com

FACS Enrichment of Zebrafish Gut Cell Suspension for scRNAseq  

Arricchimento FACS della sospensione di cellule intestinali di zebrafish per scRNAseq

Per eseguire l'ordinamento cellulare attivato dalla fluorescenza, o FACS, sulle cellule intestinali isolate dalle larve di zebrafish, utilizzare il campione di larve intere wild-type per impostare le porte della popolazione cellulare selezionata registrando 3.000 cellule sul citometro a flusso. Utilizzare un ugello da 100 micron e impostare Precisione ordinamento su Purezza a 4 vie. Mettere la provetta di raccolta contenente 200 microlitri di PBS con il 5% di siero fetale di vitello nel fax.Caricare la sospensione cellulare preparata dall'intestino isolato e, dopo aver escluso i doppietti nelle cellule morte, ordinare le singole cellule vive o DAPI-negative nella provetta di raccolta. Tenere la provetta di raccolta su ghiaccio dopo la cernita delle cellule, utilizzando un controllo di vitalità con trypan blue. Contare il numero di cellule nella sospensione con un emocitometro.Le cellule sono ora pronte per essere processate per il sequenziamento dell'RNA a singola cellula. Utilizzare circa 20.000 cellule per campione.

facs-enrichment-of-zebrafish-gut-cell-suspension-for-scrnaseq

Research

JoVE Journal

Developmental Biology

FACS Enrichment of Zebrafish Gut Cell Suspension for scRNAseq

121 Views. Per eseguire l'ordinamento cellulare attivato dalla fluorescenza, o FACS, sulle cellule intestinali isolate dalle larve di zebrafish, utilizzare il campione di larve intere wild-type per impostare le porte della popolazione cellulare selezionata registrando 3.000 cellule sul citometro a flusso. Utilizzare un ugello da 100 micron e impostare Precisione ordinamento su Purezza a 4 vie. Mettere la provetta di raccolta contenente 200 microlitri di PBS con il 5% di siero fetale di vitello nel fax.

Video: FACS Enrichment of Zebrafish Gut Cell Suspension for scRNAseq  

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.