Isolation of Undifferentiated Female Germline Cells from Adult Drosophila Ovaries

Robyn Su May Lim1, Motomi Osato2, Toshie Kai1

1 Department of Biological Sciences, National University of Singapore, 2 Cancer Science Institute of Singapore, National University of Singapore
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2E.3
DOI:  10.1002/9780470151808.sc02e03s34
Online Posting Date:  August, 2015
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Abstract

This unit describes a method for isolating undifferentiated, stem cell–like germline cells from adult Drosophila ovaries. Here, we demonstrate that this population of cells can be effectively purified from hand‐dissected ovaries in considerably large quantities. Tumor ovaries with expanded populations of undifferentiated germline cells are first removed from fly abdomens and dissociated into a cell suspension with the aid of protease treatment. The target cells, which express Vasa‐green fluorescent protein (GFP) fusion protein under the control of the germline‐specific vasa promoter, are specifically selected from the suspension via fluorescence‐activated cell sorting (FACS). These protocols can be adapted to isolate other cell types from fly ovaries, such as somatic follicle cells or escort cells, by driving GFP expression in the respective target cells. © 2015 by John Wiley & Sons, Inc.

Keywords: drosophila germline; germline stem cell; isolation; fluorescence‐activated cell sorting (FACS)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Protease Dissociation of Ovaries into Cell Suspensions
  • Support Protocol 1: Hand‐Dissection of Flies and Isolation of Ovaries
  • Support Protocol 2: Construction of 40‐μM Mesh Cell Filters
  • Basic Protocol 2: Fluorescence‐Activated Cell Sorting of Undifferentiated Germline Cells from Ovarian Cell Suspensions
  • Support Protocol 3: Fixation and Immunostaining of Germline Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Protease Dissociation of Ovaries into Cell Suspensions

  Materials
  • Isolated ovaries ( protocol 2)
  • Phosphate buffered saline (PBS; see recipe)
  • 5% (w/v) trypsin (see recipe)
  • 2.5% (w/v) collagenase (see recipe)
  • S‐FBS: Schneider's insect medium (Sigma‐Aldrich, cat. no. S0146) supplemented with 10% (v/v) fetal bovine serum (FBS), heat inactivated (Sigma‐Aldrich, cat. no. F4135)
  • Serum‐free Schneider's insect medium (Sigma‐Aldrich, cat. no. S0146)
  • 30‐μm sterile cup‐type Filcons filters (BD Biosciences, cat. no. 340625) or custom‐made 40‐μm mesh cell filters (Fig.  A, protocol 3)
  • Refrigerated microcentrifuge

Support Protocol 1: Hand‐Dissection of Flies and Isolation of Ovaries

  Materials
  • Female vasa‐GFP;bamΔ86 flies or female C587‐Gal4/+;vasa‐GFP;UAS‐Dpp/+ flies
  • Yeast paste (see recipe)
  • S‐FBS: Schneider's insect medium (Sigma‐Aldrich, cat. no. S0146) supplemented with 10% (v/v) fetal bovine serum (FBS), heat inactivated (Sigma‐Aldrich, cat. no. F4135)
  • Glass dissecting dish
  • Anesthetizing pad with carbon dioxide supply (Flystuff, cat. no. 59‐114; http://www.flystuff.com/)
  • Stereomicroscope
  • Dumont #5 forceps

Support Protocol 2: Construction of 40‐μM Mesh Cell Filters

  Materials
  • Super glue (Bostik, http://www.bostik‐us.com/)
  • 70% (v/v) ethanol
  • Penknife
  • 5‐ml serological pipet (BD Falcon, cat. no. 357543)
  • Nylon mesh with 40‐μm pore size (Millipore, cat. no. NY41)
  • Scissors

Basic Protocol 2: Fluorescence‐Activated Cell Sorting of Undifferentiated Germline Cells from Ovarian Cell Suspensions

  Materials
  • Filtered cell suspension ( protocol 1)
  • S‐FBS: Schneider's insect medium (Sigma‐Aldrich, cat. no. S0146) supplemented with 10% (v/v) fetal bovine serum (FBS), heat inactivated (Sigma‐Aldrich, cat. no. F4135)
  • Serum‐free Schneider's insect medium (Sigma‐Aldrich, cat. no. S0146)
  • 1% (w/v) (10 mg/ml) propidium iodide (PI; see recipe)
  • SPHERO Rainbow calibration particles (BD Biosciences, cat. no. 559123)
  • AccuDrop fluorescent beads (BD Biosciences, cat. no. 345249)
  • 5‐ml round bottom tubes
  • Cell sorter (BD FACSAria)

Support Protocol 3: Fixation and Immunostaining of Germline Cells

  Materials
  • S‐FBS: Schneider's insect medium (Sigma‐Aldrich, cat. no. S0146) supplemented with 10% (v/v) fetal bovine serum (FBS), heat inactivated (Sigma‐Aldrich, cat. no. F4135)
  • Cell suspension (dissociated cells or after sorting; see Basic Protocols protocol 11 and protocol 42)
  • 16% (w/v) paraformaldehyde (formaldehyde) aqueous solution (Electron Microscopy Sciences, cat. no. 30525‐89‐4)
  • Phosphate buffered saline (PBS; see recipe)
  • PBST: PBS (see recipe) containing 0.2% (v/v) Triton X‐100
  • 5% (v/v) heat‐inactivated normal goat serum (NGS; see recipe) in PBST
  • Primary antibodies:
    • Mouse anti‐GFP monoclonal 3E6 antibody at a 1:200 dilution (Invitrogen, cat. no. A‐11120)
    • Rabbit anti‐α‐Spectrin polyclonal antibody at a 1:2,500 dilution (a gift from Y. Cai, Temasek Life Sciences Laboratory, Singapore)
  • Fluorescent dye‐conjugated secondary antibodies:
    • Alexa Fluor 488‐conjugated goat anti‐mouse IgG antibody at a 1:400 dilution (Invitrogen, cat. no. A‐11029)
    • Alexa Fluor 555‐conjugated goat anti‐rabbit IgG antibody at a 1:400 dilution (Invitrogen, cat. no. A‐21429)
  • 0.2% (v/v) 4‐6‐diamidino‐2‐phenylindole (DAPI) in PBST
  • Vectashield mounting medium (Vector Laboratories, cat. no. H‐1000)
  • Refrigerated microcentrifuge
  • Positively‐charged glass slides (Fisher Scientific Fisherbrand, cat. no. 12‐550‐15)
  • Humid chamber: Petri dish, 150 mm × 15 mm, containing dampened paper towels
  • Coverslips, no. 1.5 thickness
  • Clear nail polish
  • Epifluorescence or confocal microscope
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Figures

Videos

Literature Cited

Literature Cited
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