Single and Dual Birthdating Procedures for Assessing the Response of Adult Neural Stem Cells to the Infusion of a Soluble Factor Using Halogenated Thymidine Analogs

Mireia Moreno‐Estellés1, María Díaz‐Moreno1, Pilar González‐Gómez1, Zoraida Andreu1, Helena Mira1

1 Unidad de Neurobiología Molecular, Área de Biología Celular y del Desarrollo, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.10
DOI:  10.1002/9780470151808.sc02d10s21
Online Posting Date:  May, 2012
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Abstract

The factors that regulate the switch from adult neural stem cell (aNSC) quiescence to active proliferation are poorly understood. Here we describe a method to study the in vivo effect of a soluble factor on cell cycle entry and proliferation of aNSCs located in the brain neurogenic niches. First, we provide information for implanting osmotic minipumps that will deliver the compound of interest directly into the mouse brain. When combined with the administration of the thymidine analog bromodeoxyuridine (BrdU), this technique is the most basic procedure to study the effects of a soluble factor on aNSC proliferation. We also describe a dual replication labeling protocol using two different halogenated thymidine analogs, chloro‐ and iododeoxyuridine (CldU and IdU), that allows tracking of proliferating cells and assessing cell cycle re‐entry of aNSCs at different time points. Curr. Protoc. Stem Cell Biol. 21:2D.10.1‐2D.10.20. © 2012 by John Wiley & Sons, Inc.

Keywords: neural stem cell; label‐retaining cell; IdU; CldU; BrdU; intracerebroventricular infusion; osmotic minipump

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Single Labeling of Actively Proliferating Cells to Analyze the Short‐Term Effect of a Soluble Factor on aNSCs
  • Basic Protocol 2: Dual Labeling of Two Cell Division Rounds to Analyze the Long‐Term Effect of a Soluble Factor on aNSCs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Single Labeling of Actively Proliferating Cells to Analyze the Short‐Term Effect of a Soluble Factor on aNSCs

  Materials
  • Mice: 2‐month‐old Crl:CD1(ICR) male mice (Charles River Laboratories, strain code 22)
  • Factor or drug of interest
  • Sterile saline: 0.9% (w/v) NaCl
  • 70% ethanol
  • Ketamine/xylazine anesthetic (see recipe)
  • Meloxicam analgesic (see recipe)
  • Surgical glue: Hystoacryl (B. Braun Biosurgical; http://www.bbraun.com/)
  • 10 mg/ml BrdU (see recipe for thymidine analogs)
  • IsobaVet (100% isoflurane; Schering‐Plough; store at room temperature)
  • 4% paraformaldehyde (PFA; see recipe)
  • 0.1 M PB (see recipe) with and without 0.05% sodium azide, 4°C
  • Cyanoacrylate‐based fast‐acting glue (e.g., Super Glue)
  • 2% to 3% agar (see recipe), warm
  • Cryoprotectant solution (see recipe)
  • 2 N HCl
  • 0.1 N sodium borate, pH 8.5 (see recipe)
  • Blocking solution (see recipe)
  • Primary antibody: mouse anti‐BrdU antibody (Dako, cat. no. M0744)
  • Secondary antibody: Alexa 488‐conjugated donkey anti‐mouse antibody (Invitrogen, cat. no. A21202)
  • 10 µg/ml DAPI working solution (see recipe)
  • Mowiol (see recipe)
  • Osmotic minipumps (ALZET Model 1007D for a 7‐day delivery period, 0.5 µl/hr)
  • Syringe and blunt 27‐G needle for filling minipumps (ALZET)
  • Brain infusion assembly (ALZET Brain Infusion Kit 3) including:
    • Brain infusion cannulae
    • Vinyl catheter tubes
    • Depth‐adjustment spacers
  • 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Surgery tools: large scissors, small pointed scissors, hemostatic forceps, large forceps, sterile scalpels (Fine Science Tools)
  • Micromotor drill (Leica, cat. no. 39416101)
  • Steel burr (working diameter between 0.5 and 0.8 mm; Dremel, cat. no. 26150105JA; http://www.dremel.com/)
  • Stereotaxic apparatus with mouse adaptor (Leica, cat. no. 39477001 and 39462950)
  • Animal clippers
  • Thermal blanket (Cibertec, cat. no. RTC1; http://www.cibertec.es/)
  • Airtight chamber (20 cm high, 5 cm diameter) for IsobaVet administration
  • BD Vacutainer Push Button Blood Collection Set (ref. no. 367342)
  • Peristaltic pump (Cole‐Parmer, cat. no. HV‐07524‐40)
  • 100‐mm Petri dishes
  • Vibratome (Leica VT‐1200‐S)
  • 24‐well tissue culture plates
  • Soft paint brush
  • Glass slides and cover slips
  • Confocal microscope (Leica Spectral SP5) with appropriate lasers for the fluorophores used, and camera

Basic Protocol 2: Dual Labeling of Two Cell Division Rounds to Analyze the Long‐Term Effect of a Soluble Factor on aNSCs

  Materials
  • CldU: 5‐Chloro‐2′‐deoxyuridine (see recipe for thymidine analogs)
  • IdU: 5‐Iodo‐2′‐deoxyuridine (see recipe for thymidine analogs)
  • Primary antibodies:
    • Monoclonal rat anti‐BrdU antibody (Abcam, cat. no. ab6326) for CldU detection
    • Monoclonal mouse anti‐BrdU antibody (BD, cat. no. 7580) for IdU detection
  • Secondary antibodies:
    • Alexa 488‐conjugated donkey anti‐rat (Invitrogen, cat. no. A21208)
    • Alexa 594‐conjugated donkey anti‐mouse (Invitrogen, cat. no. A21203)
  • Alcohol burner
  • Additional reagents and equipment for single labeling ( protocol 1)
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Figures

Videos

Literature Cited

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   Maslov, A.Y., Barone, T.A., Plunkett, R.J., and Pruitt, S.C. 2004. Neural stem cell detection, characterization, and age‐related changes in the subventricular zone of mice. J Neurosci. 24:1726‐1733.
   Mira, H., Andreu, Z., Suh, H., Lie, D.C., Jessberger, S., Consiglio, A., San Emeterio, J., Hortiguela, R., Marques‐Torrejon, M.A., Nakashima, K., Colak, D., Gotz, M., Farinas, I., and Gage, F.H. 2010. Signaling through BMPR‐IA regulates quiescence and long‐term activity of neural stem cells in the adult hippocampus. Cell Stem Cell 7:78‐89.
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Key References
   Mira, H., Andreu, Z., Suh, H., Lie, D.C., Jessberger, S., Consiglio, A., San Emeterio, J., Hortigüela, R., Marqués‐Torrejón, M.A., Nakashima, K., Colak, D., Götz, M., Fariñas, I., and Gage, F.H. 2010. Signaling through BMPR‐IA regulates quiescence and long‐term activity of neural stem cells in the adult hippocampus. Cell Stem Cell 7:78‐89.
  Original citation of the method described in this unit.
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