Isolating Enteric Glial Cells From the Submucosa and Lamina Propria of a Mouse

Abstract

Source: Wang, Z. et al., Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse. J. Vis. Exp. (2018).

This video demonstrates a method for isolating enteric glial cells from the submucosa and lamina propria of the mouse intestine. It outlines the steps involved in extracting submucosa and lamina propria tissue, isolating the enteric glial cells from them, and culturing the cells on a coated well-plate to establish an enteric glial cell culture.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Preparation of Sterile Poly-D-lysine (PDL) and Laminin Solutions

1. At least one day prior to cell isolation, prepare poly-D-lysine (PDL) and laminin-coated plates.
   NOTE: Both 6-well and 12-well plates were prepared depending on the experimental goals. Typically, 12-well plates were used for the quantitative analysis using western blots, whereas 6-well plates were used to hold autoclaved (sterile) coverslips for immunohistochemistry. 24-well plates were used to culture enteric....

Results

Figure 1: Set up and preparation of mouse intestines. (A) Picture of isolation set up on ice including extracted intestines. (B) Picture of 5 mL-syringe attached to a 20 G blunt end needle to flush out fecal contents. (

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Poly-D lysine (1 mg/ml stock)	Sigma	A-003-E	Dilute 1:10
Laminin (0.5 mg/ml stock)	Sigma	L4544	Dilute to 10 µg/mL on ICE
EDTA (0.5M)	Lonza	51201	Dilute 1:100 in DPBS
HEPES (1 M)	Corning	36216004	Dilute 1:100 in DPBS
Cell Recovery Solution	Corning	354253
Dulbecco's Phosphate Buffered Saline (DPBS)	HyClone	SH30028.02
DMEM/F-12	Thermo Fisher Scientific	11320033
Penicillin-Streptomycin (100X)	Life Technologies	15140-122
Gentamicin (50mg/mL stock)	Life Technologies	15750060
GDNF (10 µg stock)	Sigma	SRP3200
L-Glutamine (200 mM stock)	Life Technologies	25030-081
Nylon Mesh Celll Strainer (100 µm)	Fisher Scientific	22363549
Nylon Mesh Celll Strainer (40 µm)	Fisher Scientific	22363547
Disposable Serologic Pipet 5 ml	Fisher Scientific	13-678-11D
0.25% Trypsin-EDTA (1X)	Life Technologies	25200-056