Isolation and Culture of Primary Neurons and Glia from a Rat Urinary Bladder

Abstract

Source: Wang, R. et al., Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder. J. Vis. Exp. (2020).

This video demonstrates a technique for generating a primary culture of neurons and glia from a rat's urinary bladder. It outlines the steps involved in tissue preparation, enzymatic digestion to obtain single cells, and culturing the cells in a neuron-specific medium to establish a primary neuronal and glial culture.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of materials

1. Sterilize all instruments and ddH₂O using an autoclave before performing the experiment. Instruments include but are not limited to surgical scissors, ophthalmic scissors, forceps, spoons nucleus divider, glass dishes (60–100 mm diameter), and glass beakers.
2. Prepare the Krebs solution (Table 1): Dissolve all the chemicals with ddH₂O before use. Dissolve CaCI₂ separately and add....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin	Gibico	15050065	Enzyme digestion
48-well culture plate	Corning	3548	Coating dish
Antibiotic/antimycotic	Gibico	15240062	Culture media/Rinse media
B-27	Gibico	17504044	Culture media
BSA Fraction V	Gibico	332	Enzyme digestion
CO2 Incubator	Heraeus	B16UU	Cells culture
Collagenase type II	Sigma	2593923	Enzyme digestion
DMEM/F-12	Gibico	11330032	Rinse media
Fetal Bovine Serum	Gibico	10100147	Culture media/Rinse media
Forceps	Shanghai Jin Zhong Medical Devices	1383	10 cm; Sterile operation
Glass beakers	Huan Qiu Medical Devices	1101	50 ml; Sterile operation
Glass coverslips	WHB Scientific	WHB-48-CS	Coating dish
Glass dishes	Huan Qiu Medical Devices	1177	100 mm; Sterile operation
Laminar flow bench	Su Jie Medical Devices	CB 1400V	Sterile operation
Murine GDNF	Peprotech	AF45044	Culture media
Neurobasal-A Medium	Gibico	10888022	Culture media
Ophthalmic scissors	Shanghai Jin Zhong Medical Devices	J21010	12.5 cm; Sterile operation
Pipettes	Eppendorf	3120000240	100-1000 ul; Reagent and sample pipetting
Pipettes	Eppendorf	3120000267	10-100 ul; Reagent and sample pipetting
Poly-D-lysine	Sigma	P7280	Coating dish
Refrigerated centrifuge	Ping Fan Instrument	TGL-16A	Enzyme digestion
Shaking incubator	Haimen Kylin-Bell Lab Instruments	T8-1	Enzyme digestion
Spoons nucleus divider	Shanghai Jin Zhong Medical Devices	YZR030	12 cm; Sterile operation
Surgical scissors	Shanghai Jin Zhong Medical Devices	J21130	16 cm; Sterile operation
Surgical towel	Fu Kang Medical Devices	5002	40 x 50 cm; Sterile operation
Laminin	Sigma	L2020	Coating dish
L-glutamine	Gibico	25030081	Culture media