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Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Published: May 19th, 2013



1Molecular, Cellular and Developmental Biology, University of Michigan
* These authors contributed equally

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Flow cytometry can be used for measurements of cell viability, relative cell size, DNA content and fluorescent protein expression in live cell populations. Due to the replication of nuclear DNA during S-phase, information about DNA content in a population of cells can be used to infer relative percentages in different cell cycle phases 1-3. This method has become a cornerstone of cell cycle analysis in model systems from yeast to mammals.

The fruit fly Drosophila melanogaster has become an excellent model system for genetic in vivo analyses of cell cycle regulation. The extensive genetic tools available in flies....

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1. Fly Husbandry

  1. Cross flies of desired genotypes in narrow plastic vials with 10 ml Yeast-Glucose Medium3 or other protein-rich media of your choice. The extensive Drosophila transgenic tools available for tissue specific expression and lineage tracing with in vivo fluorescent protein expression are described in detail elsewhere4,5. Transfer parents to fresh vials daily to obtain series of 24 hr progeny collections, or for more precise staging, collect embryos on agar .......

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Figure 2 shows representative results for a larval wing sample, expressing GFP in the posterior half of the tissue, using the provided GFP template. Similar results are obtained with the same tissue type and expression pattern for RFP using the provided RFP template (Figure 3A). The provided templates and voltages (Table 2) are suitable for analysis of larval eyes (Figure 3B), brains and wings, as well as pupal eyes, brains (Figure 3D) and wings. H.......

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The protocol described here allows for analysis of cell cycle, relative cell size and relative cell number in live Drosophila tissues at various developmental stages. When this analysis is coupled with cell-type specific fluorescent protein expression or lineage tracing, detailed information can be obtained about cellular responses to discreet cell cycle or growth perturbations. As proof of principle, we disrupted quiescence in the pupal fly brain by expressing G1-S cell cycle regulators in GFP labeled cel.......

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We thank Aida de la Cruz for developing and teaching the original protocol on which this version is based10. Work in the Buttitta Lab is supported by NIH grant GM086517.


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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
12x75 mm Polystyrene Round-Bottom 5 ml Test Tube BD Falcon 352058 5 ml tubes
Attune Acoustic Focusing Cytometer Life Technologies/ Applied Biosystems 4445315 Blue / Violet configuration
Attune Cytometer Software (version 1.2.5) Life Technologies/ Applied Biosystems Free PC only
Attune Performance Tracking Beads (5 x 106 beads/ ml) Life Technologies/ Applied Biosystems 4449754 For daily performance test
Dumont #5 Inox forceps Fine Science Tools 11251-20
Embryo dishes 30 mm x 12mm Electron Microscopy Sciences 70543-30 Glass dissection dishes
Eppendorf Thermomixer Eppendorf 022670051
Trypsin-EDTA Solution (10x) Sigma T4174
Vannas-Tübingen Spring Scissors Fine Science Tools 15003-08 Straight 5mm Cutting Edge
Vybrant DyeCycle Violet Stain Life Technologies/ Invitrogen V35003
Table 1. Required reagents and instruments.

Live DNA Stain Solution (10 ml):

1 ml 10X Ca2+ Mg2+ free PBS (pH7.2)
9 ml 10X Trypsin-EDTA (Sigma)
5 μl Invitrogen Vybrant DyeCycle Violet (note that this is 0.25X the recommended concentration for mammalian cells. We find that higher concentrations are toxic to Drosophila cells.)

10X Ca2+ Mg2+ free PBS (pH7.2): 1.37M NaCl, 27 mM KCl, 100mM Na2HPO4 (dibasic), 20mM KH2PO4 (monobasic) adjusted to pH 7.2

Threshold (x1000) 100 10 10 10
Voltage (mV) 2950 4250 1800 1150

Table 2. Threshold and voltage setting for the analysis in Figure 2.

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