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Preparation of Primary Neurons from Rat Median Preoptic Nucleus (MnPO)

从大鼠正中视前核(MnPO)中制备原代神经元

Emmanuelle Berret Emmanuelle Berret

发布: 2013年12月05日第3卷第23期 DOI: 10.21769/BioProtoc.984 浏览次数: 10104

评审: Xuecai Ge

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Cover of The Journal of Neuroscience, featuring study using the protocol.

Feb 2013

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Abstract

Studying cell physiology is really important to understand their function and to determine action mechanisms taking place in these cells. Using brain slices can be sometime difficult to directly access to cells like neurons. Even if it more steps are needed to get cultured cells, this type of preparation allow a better access to neurons and provide a good way to study their basal properties. Here we describe a protocol of a short term primary neurons culture from MnPO, allowing to kept neurons in good condition to perform electrophysiological recordings.

Materials and Reagents

  1. Wistar rats (3 weeks old) (Charles River Laboratories International)
  2. Ketamine-xylasine mixture
  3. Pronase (Sigma-Aldrich, catalog number: 9036-06-0 )
  4. Thermolysin (Sigma-Aldrich, catalog number: 9073-78-3 )
  5. Bovine Serum Albumin (BSA) (Life Technologies, catalog number: 15561020 )
  6. Laminin (Sigma-Aldrich, catalog number: 114956-81-9 )
  7. Ruthenium red
  8. KCl (Sigma-Aldrich, catalogue number: 7447-40-7 )
  9. CaCl2 (Sigma-Aldrich, catalogue number: 10043-52-4 )
  10. MgCl2 (Sigma-Aldrich, catalogue number: 7786-30-3 )
  11. NaHCO3 (Sigma-Aldrich, catalogue number: 144-55-8 )
  12. NaH2PO4 (Sigma-Aldrich, catalogue number: 7558-80-7 )
  13. NaCl (Sigma-Aldrich, catalogue number: 7647-14-5 )
  14. Sucrose (Sigma-Aldrich, catalogue number: 57-50-1 )
  15. HEPES (Sigma-Aldrich, catalogue number: 7365-45-9 )
  16. D-Glucose (Sigma-Aldrich, catalogue number: 50-99-7 )
  17. Dissection solution (see Recipes)
  18. Artificial Cerebrospinal Fluid (aCSF) (see Recipes)

Equipment

  1. Bath
  2. Vibratome
  3. 0.45 μm sterilize filter
  4. 350 μm Sagittal slice containing MnPO
  5. Needle
  6. Pasteur pipettes
  7. Centrifuge
  8. 37 °C, 95% O2-5% CO2 Cell culture incubator
  9. 6 hole petri dish (Thermo Fisher Scientific, catalog number: 50-341-84 )
  10. Micro-cover glasses (Thermo Fisher Scientific, catalogue number: NC9216450 )
  11. 15 ml Falcon tubes
  12. Oxygen and Carbogen tank
  13. Binocular

Procedure

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Berret, E. (2013). Preparation of Primary Neurons from Rat Median Preoptic Nucleus (MnPO). Bio-protocol 3(23): e984. DOI: 10.21769/BioProtoc.984.
  2. Berret, E., Nehme, B., Henry, M., Toth, K., Drolet, G. and Mouginot, D. (2013). Regulation of central Na+ detection requires the cooperative action of the NaX channel and α1 Isoform of Na+/K+-ATPase in the Na+-sensor neuronal population. J Neurosci 33(7): 3067-3078.

    3. ris ris Download Citation in RIS Format

分类

神经科学 > 细胞机理 > 细胞分离和培养

细胞生物学 > 细胞分离和培养 > 细胞分离

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