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. 2020 Oct 20;1(3):100144.
doi: 10.1016/j.xpro.2020.100144. eCollection 2020 Dec 18.

Preparing Highly Viable Single-Cell Suspensions from Mouse Pancreatic Islets for Single-Cell RNA Sequencing

Hugo Lee  1 , Feyza Engin  1   2
Affiliations

Preparing Highly Viable Single-Cell Suspensions from Mouse Pancreatic Islets for Single-Cell RNA Sequencing

Hugo Lee et al. STAR Protoc. .

Abstract

Pancreatic islets consist of several cell types, including alpha, beta, delta, epsilon, and PP cells. Due to cellular heterogeneity, it is challenging to interpret whole-islet transcriptome data. Single-cell transcriptomics offers a powerful method for investigating gene expression at the single-cell level and identifying cellular heterogeneity and subpopulations. Here, we describe a protocol for mouse pancreatic islet isolation, culturing, and dissociation into a single-cell suspension. This protocol yields highly viable cells for successful library preparation and single-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020).

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Inflation of Pancreas (A) Close-up image of the bile duct. The incision point for needle insertion is denoted by a blue circle. (B) The abdominal cavity showing the pancreas before inflation. (C) The inflated pancreas after collagenase injection through the bile duct. BD, bile duct; GB, gallbladder; IP, incision point; P, pancreas; SO, sphincter of Oddi.
Figure 2
Figure 2
Detaching Pancreas from Surrounding Organs Black arrows indicate the points to detach between the pancreas and (A) stomach or (B) large intestine. LI, large intestine; P, pancreas; S, stomach.
Figure 3
Figure 3
Microscopic Evaluation of Isolated Islets Representative image of isolated islets of varying size from a 4-week-old female NOD mouse (A) before and (B) after handpicking. Images were taken under a stereo microscope with bottom lighting at 20× magnification.
Figure 4
Figure 4
Representative Image of Cell Viability Assessed by an Automated Cell Counter Measurement of cell viability following dissociation of pancreatic islets into a single-cell suspension was performed by mixing the cell suspension with trypan blue at a 1:1 ratio before being loaded onto the Countess II automated cell counter. Cells outlined in green are live, while cells outlined in red are dead.
See this image and copyright information in PMC

References

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