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

Summary

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Abstract

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague–Dawley rats. In this method, the neurons and glia were incubated in a 37 °C, 5% CO₂ incubator for 5–7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by β-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

Introduction

The lower urinary tract has two main functions: periodic urine storage and micturition¹. The lower urinary tract nervous system (LUTNS) controls these functions and is delicate and susceptible to many neuropathies, which can be innate (porphyria), acquired (Lyme disease), secondary to disease states (diabetic cystopathy), drug induced (hemorrhagic cystitis), surgery caused (abdominoperineal resection), or injury caused (traumatic spinal cord injury)^2,3,4,5,6,

Protocol

All experimental protocols and animal procedures complied with the ethical principle guidelines of the National Research Council.

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 in diameter), and glass breakers.
2. Prepare the Krebs solution as follows .......

Discussion

Plate Preparation
The use of glass coverslips in 6-, 12-, or 48-well culture plates for immunofluorescent or calcium imaging experiments is an economical and sample-sparing operation. Cells grow well in plates without coverslips during the preparation of primary cell cultures. Therefore, coverslips are dispensable in experiments, such as Western blot or polymerase chain reaction. Furthermore, coating is a necessary step before plating cells, with or without coverslips. Laminin and poly-D-lysine are.......

Materials

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
Anti-Glial Fibrillary Acidic Protein Antibody	Santa Cruz	sc-33673	ICC
B-27	Gibico	17504044	Culture media
BSA Fraction V	Gibico	332	Enzyme digestion
Choline Acetyltransferase Antibody	Abcam	ab18736	ICC
CO2 Incubator	Heraeus	B16UU	Cells culture
Collagenase type II	Sigma	2593923	Enzyme digestion
DMEM/F-12	Gibico	11330032	Rinse media
DYNLL2 Antibody	Santa Cruz	sc-13969	ICC
Fetal Bovine Serum	Gibico	10100147	Culture media/Rinse media
Forceps	Shanghai Jin Zhong Medical Devices	1383	10 cm; Sterile operation
Glass breakers	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
Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488	Southernbiotech	3050-30	ICC
Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555	Southernbiotech	3050-30	ICC
Hoechst 33342	BD	561908	ICC
Laminar flow bench	Su Jie Medical Devices	CB 1400V	Sterile operation
Laminin	Sigma	L2020	Coating dish
L-glutamine	Gibico	25030081	Culture media
MAP-2 Antibody	Affinity	AF5156	ICC
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
SLC17A9 Antibody	MBL International	BMP079	ICC
Spoons nucleus divider	Shanghai Jin Zhong Medical Devices	YZR030	12 cm; Sterile operation
Substance P Antibody	Santa Cruz	sc-58591	ICC
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
Synapsin-1 Antibody	CST	5297T	ICC
Tubulin beta Antibody(β-III-tubulin)	Affinity	AF7011	ICC