Improve Research Reproducibility A Bio-protocol resource
  • search
  • 提交稿件
  • 订阅
  • CN change language
该实验方案已做出更正， 查看更正通知。
bpdetail_icon_lock
Peer-reviewed

Establishment of an in vitro Differentiation and Dedifferentiation System of Rat Schwann Cells

大鼠雪旺细胞体外分化和去分化体系的建立

YZ Ying Zou

发布: 2023年03月05日第13卷第5期 DOI: 10.21769/BioProtoc.4631 浏览次数: 1134

评审: Gal HaimovichPhilipp WörsdörferAnonymous reviewer(s)

download pdf 下载 PDF 下载 PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Molecular Neurobiology, featuring study using the protocol.

Jan 2022

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

人诱导多能干细胞来源心肌细胞的分化、维持及收缩特性分析

人诱导多能干细胞来源心肌细胞的分化、维持及收缩特性分析

Matthijs Snelders [...] Jeroen Essers

2025年03月05日 2357 阅读

雪貂气道干细胞的分离与培养

雪貂气道干细胞的分离与培养

Ziying Yan [...] Feng Yuan

2025年07月20日 994 阅读

基于ImmunoCellCycle-ID方法精准识别RPE1细胞的细胞周期阶段

基于ImmunoCellCycle-ID方法精准识别RPE1细胞的细胞周期阶段

Syon Reddy [...] Aussie Suzuki

2025年08月05日 921 阅读

Abstract

In the peripheral nervous system, Schwann cells are the primary type of glia. This protocol describes an in vitro differentiation and dedifferentiation system for rat Schwann cells. These cultures and systems can be used to investigate the morphological and biochemical effects of pharmacological intervention or lentivirus-mediated gene transfer on the process of Schwann cell differentiation or dedifferentiation.


Graphical abstract


Keywords: Peripheral nerve search Schwann cells search Differentiation search Dedifferentiation search In vitro search

Background

Schwann cells are the primary glial cells of the peripheral nervous system. During axonal sorting and myelination in the peripheral nerves, Schwann cells originate from neural crest cells that differentiate into mature phenotypes (Cristobal and Lee, 2022). Moreover, Schwann cells’ incredible plasticity is one of the most important characteristics following nerve damage or demyelination (Nocera and Jacob, 2020). Schwann cells undergo dedifferentiation after injury and redifferentiate to promote nerve regeneration and complete functional recovery (Jessen and Mirsky, 2019). Therefore, it is important to study Schwann cells’ differentiation and dedifferentiation status to understand their role in nerve development and injury. As reported previously, dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) induces Schwann cells to acquire a differentiated phenotype (Yamauchi et al., 2011). After stimulation with 1 mM dbcAMP, Schwann cells from rats change from bipolar or tripolar to flat within 24 h. By contrast, mouse Schwann cells retain their bipolar or tripolar morphology after dbcAMP treatment (Arthur-Farraj et al., 2011). And a previous study described in vitro assays of scalable differentiation and dedifferentiation of Schwann cell in the absence of neurons (Monje PV., 2018). Based on previous studies, we established a detailed and simplified system for differentiation and dedifferentiation of Schwann cells. In order to clearly distinguish morphological changes, we performed the differentiation and dedifferentiation assay using rat Schwann cells. In brief, the first step was to obtain Schwann cells from the spinal nerves of rats based on a previous study (Wen et al., 2017). During the second step, differentiation and dedifferentiation assays were conducted. Schwann cells were purified and passaged, and experiments were conducted on the third passage. Finally, Schwann cell status was determined by morphological examination, western blotting analysis, and immunofluorescence detection.

Materials and reagents

  1. Cell culture dish (3.5 and 6 cm) (JET BIOFILT, catalog numbers: CD000035, TCD-010-060)

  2. 50 mL centrifuge tubes (Corning, catalog number: 430828)

  3. 15 mL centrifuge tubes (Corning, catalog number: 430790)

  4. 1.5 mL tube (JET BIOFILT, catalog number: CFT000015)

  5. Polyvinylidene fluoride membrane (PVDF) (Bio-Rad, catalog number: 1620177)

  6. Cell glass coverslips (diameter: 12 mm, thickness: 0.13–0.17 mm) (Fisherbrand, catalog number: FIS12-545-80)

  7. Neonatal Sprague-Dawley (SD) rat [postnatal 1–2 days (P1–P2)]

  8. Distilled water

  9. 75% ethanol

  10. Phosphate buffered saline (PBS) (Gibco, catalog number: 10010023)

  11. Poly-L-lysine hydrobromide (PLL) (Sigma-Aldrich, catalog number: P1274)

  12. 0.25% trypsin-EDTA (Gibco, catalog number: 25200072)

  13. Fetal bovine serum (FBS) (Corning, catalog number: 35-076-CV)

  14. DMEM/F12 (Gibco, catalog number: 11330057)

  15. Cytosine arabinoside (Ara-C) (Sigma-Aldrich, catalog number: C1768)

  16. Recombinant human heregulin β-1 (PeproTech, catalog number: 100-03)

  17. Forskolin (Sigma-Aldrich, catalog number: F6886)

  18. 4% paraformaldehyde (PFA) (Biosharp, catalog number: BL539A)

  19. Dimethyl sulfoxide (DMSO), suitable for cell culture (Beyotime, catalog number: ST038)

  20. Dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) (Sigma-Aldrich, catalog number: D0627)

  21. Triton X-100 (Sigma-Aldrich, catalog number: V900502)

  22. Tween-20 (Sigma-Aldrich, catalog number: P1379)

  23. Phalloidin (Abcam, catalog number: ab176759)

  24. RIPA lysis buffer (FUDE Biological Technology, catalog number: FD009)

  25. Rabbit monoclonal (EP1039Y) anti-p75 (Abcam, catalog number: ab52987)

  26. Rabbit anti-Krox20 (Novus Biologicals, catalog number: 13491-1-AP)

  27. Mouse monoclonal anti-c-Jun (BD Biosciences, catalog number: 610326)

  28. Mouse monoclonal (9-9-3) anti-Sox2 (Abcam, catalog number: ab79351)

  29. Alexa Fluor® 488 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, catalog number: A11001)

  30. Alexa Fluor® 568 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, catalog number: A11011)

  31. 4’,6-diamidino-2’-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D8417)

  32. Penicillin–streptomycin (Gibco, catalog number: 15140-122)

  33. Omni-ECLTM Light Chemiluminescence kit (EpiZyme, catalog number: SQ201)

  34. 10% dodecyl sulfate sodium salt-polyacrylamide gel (EpiZyme, catalog number: PG112)

  35. 5% non-fat milk (Solarbio, catalog number: D8340)

  36. Tris-buffered saline (Sigma-Aldrich, catalog number: 93318)

  37. Horseradish peroxidase (HRP)-conjugated secondary antibody (Invitrogen, catalog numbers: 31430, 31460)

  38. 1,000× Ara-C (10 mM in distilled water, see Recipes)

  39. 1,000× dbcAMP (1,000 mM in DMSO, see Recipes)

  40. 30 mM forskolin stock solution (see Recipes)

  41. Complete growth medium of rat Schwann cells (see Recipes)

  42. 10% FBS (see Recipes)

  43. 3% FBS (see Recipes)

  44. 1% FBS (see Recipes)

  45. 1 mM dbcAMP (see Recipes)

  46. 0.1% Triton X-100 (see Recipes)

  47. 5% gelatin (see Recipes)

  48. PBST (see Recipes)

  49. Blocking buffer (see Recipes)

Equipment

  1. Pipettes (Thermo Fisher Scientific, 10, 200, and 1,000 μL)

  2. CO2 incubator (Thermo Fisher Scientific, model: Heracell 150i)

  3. Surgical scissors and forceps (Shenzhen RWD, catalog numbers: S14014 and F12029-09)

  4. Spring scissors (Shenzhen RWD, catalog number: S11001)

  5. Superfine forceps (Shenzhen RWD, catalog number: F13002)

  6. Stereomicroscope (Jiangnan Novel, model: SZ6060)

  7. Centrifuge (Eppendorf, model: Micro21)

  8. Phase contrast microscope (Zeiss, model: Primovert)

  9. Fluorescence microscope (Zeiss, model: Axio Imager A2)

Software

  1. ImageJ (Version 1.8.0, https://imagej.en.softonic.com/)

Procedure

English
中文翻译

文章信息

版权信息

© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).

如何引用

Ying, Z. (2023). Establishment of an in vitro Differentiation and Dedifferentiation System of Rat Schwann Cells. Bio-protocol 13(5): e4631. DOI: 10.21769/BioProtoc.4631.

ris ris Download Citation in RIS Format

分类

神经科学 > 周围神经系统 > 施万细胞

细胞生物学 > 细胞分离和培养 > 细胞分化

细胞生物学 > 细胞成像 > 固定细胞成像

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件