Evaluation of Proliferation of Neural Stem Cells In Vitro and In Vivo

Maria Inês Morte1, Bruno P. Carreira1, Vanessa Machado2, Anália Carmo3, Isabel Nunes‐Correia4, Caetana M. Carvalho5, Inês M. Araújo2

1 These authors contributed equally to this work., 2 IBB‐Institute for Biotechnology and Bioengineering, Centre of Molecular and Structural Biomedicine, University of Algarve, Faro, Portugal, 3 Centre for Neuroscience and Cell Biology, Cellular Immunology and Oncobiology Group, University of Coimbra, Coimbra, Portugal, 4 Centre for Neuroscience and Cell Biology, Flow Cytometry Unit, University of Coimbra, Coimbra, Portugal, 5 Centre for Neuroscience and Cell Biology, Neuroendocrinology and Neurogenesis Group, University of Coimbra, Coimbra, Portugal
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.14
DOI:  10.1002/9780470151808.sc02d14s24
Online Posting Date:  February, 2013
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Abstract

This unit describes two basic protocols for the detection of the proliferation of neural stem cells (NSC). The first one addresses cell proliferation in cultures, starting with primary cell cultures isolated from the mouse subventricular zone (SVZ), in which SVZ‐derived NSC are kept in culture as neurospheres. By using this culture system, we are able to study different stages of adult neurogenesis, such as proliferation, differentiation, migration, and survival. Thus, in the first basic protocol, we describe two different techniques to evaluate cell proliferation based on EdU incorporation: (a) immunocytochemistry and (b) flow cytometry. EdU, a new thymidine analog, which is detected by a reproducible and sensitive method based on click chemistry, does not require DNA denaturation, as is the case with BrdU. Thus, co‐labeling of EdU with other specific antibodies of extracellular or intracellular targets, as well as other DNA dyes, is possible. In the second basic protocol, we describe an in vivo assay to evaluate proliferation of NSC in the dentate gyrus of hippocampus of adult mice, by both BrdU and EdU detection. With this approach, it is also possible to study different stages of adult neurogenesis, by co‐labeling thymidine analogs with other specific markers, such as doublecortin (DCX) or neuronal nuclei protein (NeuN). Curr. Protoc. Stem Cell Biol. 24:2D.14.1‐2D.14.24. © 2013 by John Wiley & Sons, Inc.

Keywords: EdU; BrdU; proliferation; subventricular zone; hippocampus

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Evaluation of Cell Proliferation in SVZ Cultures
  • Support Protocol 1: Fixation of SVZ‐Derived NSC Cultures with PFA
  • Basic Protocol 2: EdU Detection by Fluorescence Microscopy
  • Alternate Protocol 1: EdU Detection by Flow Cytometry
  • Support Protocol 2: Preparation of Gelatin‐Coated Slides
  • Basic Protocol 3: Evaluation of Cell Proliferation in Brain Tissue
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Evaluation of Cell Proliferation in SVZ Cultures

  Materials
  • Animals: C57BL/6J mouse pups (P0 to P3; Charles River Laboratories, http://www.criver.com)
  • Dissection medium: Hank's balanced salt solution (HBSS; Invitrogen, cat. no. 14170‐088) plus 0.25% (v/v) gentamicin (Invitrogen, cat. no. 15750‐060)
  • SVZ culture medium (see recipe)
  • Dulbecco's Modified Eagle's Medium:F‐12 nutrient mixture (DMEM/F12) with GlutaMAX‐I (Invitrogen, cat. no. 10565‐018; optional)
  • B‐27 serum‐free supplement (50×), liquid (Invitrogen, cat. no. 17504‐044; optional)
  • Antibiotic (10,000 U/ml penicillin, 10,000 U/ml streptomycin; Invitrogen, cat. no. 15140‐130)
  • 0.25% trypsin/0.265 mM EDTA (1×) with phenol red (Invitrogen, cat. no. 25200‐056)
  • Hanks' balanced salt solution (HBSS; Invitrogen, cat. no. 14170‐088)
  • 0.04% (w/v) trypan blue
  • DNase I (Sigma‐Aldrich, cat. no. DN25)
  • Bovine serum albumin (BSA)
  • Recombinant murine epidermal growth factor (EGF; Peprotech, cat. no. 315‐09)
  • Recombinant human basic fibroblast growth factor (bFGF; Peprotech, cat. no. 100‐18B)
  • 10 mg/ml 5‐ethynyl‐2′‐deoxyuridine (EdU; Invitrogen, cat. no. A10044) stock
  • 4% (w/v) paraformaldehyde/4% (w/v) sucrose (PFA/Suc) in 0.01 M phosphate‐buffered saline (PBS; Invitrogen, cat. no. 7011‐036)
  • 70% (v/v) ethanol
  • Tools: fine‐tipped (or small pointed) scissors, fine‐tipped forceps, and fine‐curved forceps (Fine Science Tools, http://www.finescience.de/); steel brain matrix for mice, 1.0 mm, coronal, 40 to 75 g (Stoelting, cat. no. 51386; https://www.stoeltingeurope.com/); single‐edge razor blades (Stoelting, cat. no. 52177; https://www.stoeltingeurope.com/)
  • Petri dishes (60 mm)
  • 15‐ml and 50‐ml conical centrifuge tubes (sterile; e.g., BD Falcon)
  • Stereomicroscope (e.g., Zeiss Stemi DV4 Stereo Microscope, cat. no. NT55‐594)
  • Hemacytometer
  • Tissue culture Petri dishes (60 mm)
  • 1.5 and 0.5‐ml microcentrifuge tubes
  • Centrifuge
  • Uncoated 12‐well plates
  • 16‐mm‐diameter glass coverslips coated with poly‐L‐lysine by immersing in 0.1 mg/ml poly‐L‐lysine 2 hr at room temperature in the flow chamber
  • 12‐well plates coated with poly‐L‐lysine by filling the wells with 0.1 mg/ml poly‐L‐lysine 2 hr at room temperature in the flow chamber
NOTE: All procedures with live cells are performed in a Class II biological hazard flow chamber, using sterile equipment and solutions and proper aseptic technique.NOTE: All incubations are performed in a 95% air/5% CO 2 humidified incubator at 37°C unless otherwise noted.

Support Protocol 1: Fixation of SVZ‐Derived NSC Cultures with PFA

  Materials
  • SVZ‐derived NSC cultures plated in glass coverslips coated with poly‐L‐lysine, drug‐treated and EdU‐incubated ( protocol 1, step 22)
  • 0.01 M phosphate‐buffered saline (PBS; Invitrogen, cat. no. 7011‐036)
  • 4% paraformaldehyde/4% sucrose (PFA‐suc) (w/v) in PBS (see recipe)
  • Plastic Pasteur pipets
  • Vacuum aspirator

Basic Protocol 2: EdU Detection by Fluorescence Microscopy

  Materials
  • SVZ‐derived NSC cultures, drug‐treated and EdU‐incubated, fixed with 4% PFA/4% sucrose ( protocol 1, step 22 and protocol 2)
  • 3% (w/v) bovine serum albumin (BSA) (Merck Millipore, cat. no. 112018) in 0.01 M phosphate‐buffered saline (PBS)
  • 0.5% (v/v) Triton X‐100 in 0.01 M phosphate‐buffered saline (PBS)
  • Click‐iT EdU Alexa Fluor 488 HCS Assay kit (Invitrogen, cat. no. C10350)
  • 10 mg/ml Hoechst 33342 stock solution (Invitrogen, cat. no. H3570)
  • 0.01 M phosphate‐buffered saline (PBS; Invitrogen, cat. no. 7011‐036)
  • DAKO fluorescence mounting medium (DAKO, cat. no. S3023; http://www.dako.com/)
  • Clear nail polish
  • Orbital shaker
  • Plastic Pasteur pipets
  • Vacuum aspirator
  • Microscopy glass slides
  • Fluorescence microscope
  • Additional reagents and equipment for immunohistochemistry (Hofman, )

Alternate Protocol 1: EdU Detection by Flow Cytometry

  Materials
  • SVZ‐derived NSC cultures, drug‐treated and EdU‐incubated ( protocol 1, step 21) in gelatin‐coated 12‐well plates
  • 0.01 M phosphate‐buffered saline (PBS; Invitrogen, cat. no. 7011‐036)
  • StemPro Accutase cell dissociation reagent (Invitrogen, cat. no. A11105‐01)
  • 70% (v/v) ethanol
  • Click‐iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen, cat. no. C‐10425)
  • 20 mg/ml ribonuclease A stock (RNase A; Invitrogen, cat. no. 12091‐021)
  • 7‐AAD (Invitrogen, cat. no. C35002)
  • 12 × 75–mm FACS tubes (BD Falcon, cat. no. 35‐2008)
  • Centrifuge
  • Flow cytometer with a 488 nm argon‐ion laser and the appropriate filter sets for collecting fluorescence emissions from Alexa Fluor 488 and 7‐AAD (detection with a 530/30 nm bandpass and 670 nm long pass filter, respectively
  • Additional reagents and equipment for flow cytometry (Coligan et al., , Chapter 5)

Support Protocol 2: Preparation of Gelatin‐Coated Slides

  Materials
  • Gelatin powder
  • Chromium potassium sulfate dodecahydrate
  • Magnetic stirrer and stir bar
  • Filter paper
  • Glass slides
  • 65°C oven

Basic Protocol 3: Evaluation of Cell Proliferation in Brain Tissue

  Materials
  • Brain sections, cryosectioned, obtained by transcardiac perfusion of fixative
  • 0.01 M phosphate‐buffered saline (PBS; Invitrogen, cat. no. 7011‐036)
  • 1 M HCl
  • 0.25% (v/v) Triton X‐100 in 0.01 M PBS
  • Blocking solution: 5% (v/v) normal goat serum (NGS; Vector Laboratories, cat. no. S‐1000) or 5% normal horse serum (NHS; Vector Laboratories, cat. no. S‐2000), depending on species from which primary antibody was derived, in 0.25% (v/v) Triton X‐100/0.01 M PBS
  • Primary antibodies: rat anti‐BrdU (AbDSerotec, cat. no. MCA2060), mouse anti‐NeuN (Chemicon, cat. no. MAB377), and goat anti‐DCX (Santa Cruz Biotechnology, cat. no. sc‐8066)
  • Secondary antibodies: goat or donkey anti–rat IgG conjugated with Alexa Fluor 488 (Invitrogen, cat. no. A‐11006 and cat. no. A‐21209, respectively); goat anti–mouse IgG conjugated with Alexa Fluor 594 (Invitrogen, cat. no. A‐11005); donkey anti–goat IgG conjugated with Alexa Fluor 594 (Invitrogen, cat. no. A‐11058)
  • 3% (w/v) bovine serum albumin (BSA) in 0.01 M PBS
  • 0.5% (v/v) Triton X‐100 in PBS
  • Click‐iT EdU Alexa Fluor 488 HCS Assay kit (Invitrogen, cat. no. C10350)
  • 2 µg/ml Hoechst 33342 in 0.01 M PBS
  • Orbital shaker
  • Glass vials
  • Small paintbrush
  • 65°C oven
  • 2% gelatin–coated slides ( protocol 5)
  • Aluminum foil
  • Petri dish
  • Coverslips
  • DAKO Fluorescence Mounting Medium (DAKO, cat. no. S3023)
  • Fluorescence microscope
NOTE: All incubations and rinsing steps are made on an orbital shaker, at room temperature.
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Figures

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Literature Cited

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