Time‐Lapse Live Imaging of Stem Cells in Drosophila Testis

Jun Cheng1, Alan J. Hunt1

1 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2E.2
DOI:  10.1002/9780470151808.sc02e02s11
Online Posting Date:  November, 2009
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Abstract

This unit describes a protocol for time‐lapse live‐imaging of stem cells in Drosophila testis. Testis tips are dissected from Drosophila, sliced, and transferred to glass‐bottom chambers where the stem cells residing in their native microenvironment can be monitored in real time. This protocol, facilitated with various fluorescence‐labeled markers, allows dynamic cellular processes in stem cells to be characterized throughout the cell cycle. Curr. Protoc. Stem Cell Biol. 11:2E.2.1‐2E.2.8. © 2009 by John Wiley & Sons, Inc.

Keywords: Drosophila gonad; stem cell; tissue culture; time‐lapse live imaging; epifluorescence microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Time‐Lapse Live Imaging of Drosophila Testes
  • Support Protocol 1: Dissecting and Tissue‐Culturing Drosophila Testes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Time‐Lapse Live Imaging of Drosophila Testes

  Materials
  • Culture of Drosophila testes tips in petri dishes ( protocol 2)
  • A high‐quality inverted microscope with epifluorescence capability (e.g., Zeiss Axiovert 200)
  • 3‐axis computer‐controlled microscope stage (e.g., Madcity Labs)
  • Automated shutter in the epifluorescence light path (e.g., Uniblitz)
  • Plan‐NEOFLUAR 40× objective with NA = 0.75 or AchroPlan 63× objective with NA = 0.8
  • A highly sensitive CCD camera [e.g., Hamamatsu Electron multiplier (EM) CCD camera]
  • Computer with software for controlling shutter, specimen stage, and image acquisition (e.g., Metamorph or ImageJ)
NOTE: 40× NA = 0.75 and 63× NA = 0.8 objectives provide the necessary depth of field while maintaining sufficient resolution and epifluorescence intensity.NOTE: To prevent any photo‐bleaching and potential photodamage to the testis tissue, exposure time is minimized. To achieve this goal, a highly sensitive CCD camera and a fast‐response shutter in the epifluorescence light pathway are necessary. Moreover, software control of the shutter and CCD camera is required to synchronize the shutter open/close with the timing of the image acquisition.

Support Protocol 1: Dissecting and Tissue‐Culturing Drosophila Testes

  Materials
  • 70% ethanol
  • Drosophila culture medium (DCM; see recipe)
  • Drosophila male flies
  • Regenerated cellulose membrane (Spectrum Lab)
  • Carbon dioxide flowbed (Genesee Scientific)
  • Stereomicroscope (Leica)
  • Standard dissecting equipment including:
    • Forceps (Dumont #5)
    • Scalpel (Feather #15)
    • Scissors
  • 35‐mm glass‐bottom Petri dish with 20‐mm microwell (MatTek)
  • Parafilm
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Figures

Videos

Literature Cited

Literature Cited
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