facs enrichment of zebrafish gut cell suspension for scrnaseq

Procesamiento de células intestinales de larvas de pez cebra para la secuenciación de ARN unicelular

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).

Autor destacado: Aislamiento y análisis de células intestinales de larvas de pez cebra para investigar los aspectos transcriptómicos del desarrollo gastrointestinal 

Aislamiento intestinal de larvas de pez cebra para la secuenciación de ARN de una sola célula

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  

FACS Enriquecimiento de la suspensión de células intestinales de pez cebra para scRNAseq

Para realizar la clasificación celular activada por fluorescencia, o FACS, en las células intestinales aisladas de larvas de pez cebra, utilice la muestra de larvas enteras de tipo salvaje para establecer las puertas de la población celular clasificada mediante el registro de 3.000 células en el citómetro de flujo. Use una boquilla de 100 micras y establezca Precisión de clasificación en Pureza de 4 vías. Coloque el tubo de recolección que contiene 200 microlitros de PBS con un 5% de suero fetal para terneros en la máquina de fax.Cargue la suspensión celular preparada a partir de los intestinos aislados y, después de excluir los dobletes en las células muertas, clasifique las células individuales vivas o negativas para DAPI en el tubo de recolección. Mantenga el tubo de recolección en hielo después de la clasificación de las células, empleando una verificación de viabilidad con azul de tripano. Cuente el número de células en la suspensión con un hemocitómetro.Las células ya están listas para ser procesadas para la secuenciación de ARN de una sola célula. Utilice aproximadamente 20.000 células por muestra.

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

Research

JoVE Journal

Developmental Biology

FACS Enrichment of Zebrafish Gut Cell Suspension for scRNAseq

121 vistas. Para realizar la clasificación celular activada por fluorescencia, o FACS, en las células intestinales aisladas de larvas de pez cebra, utilice la muestra de larvas enteras de tipo salvaje para establecer las puertas de la población celular clasificada mediante el registro de 3.000 células en el citómetro de flujo. Use una boquilla de 100 micras y establezca Precisión de clasificación en Pureza de 4 vías. Coloque el tubo de recolección que contiene 200 microlitros de PBS con un 5% de...

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.