This work was supported by the division of intramural research at the National Institutes of Health/NHLBI (1ZIAHL006126) and a Lenfant Biomedical Postdoctoral Fellowship awarded to DAL.

1. Preparation of Materials Required to Explant Third Instar Larval Brains
<ol>
	<li>Prepare tools required for microdissection.
	<ol>
		<li>Forge two dissecting tools (a scalpel and a hook) by first placing a single dissecting pin into each pin holder. Secure the pins tightly by hand or with pliers (Figure 1C).</li>
		<li>Use a pair of old forceps to bend one dissecting pin to an approximately 120&#176; angle. Do this under a dissecting microscope to ensure that the top half of the pin lies flat when ...

<ol>
	<li>Truman, J. W., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+patterns+of+neurogenesis+in+the+central+nervous+system+of+Drosophila+melanogaster.">Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster.</a> Developmental Biology. 125, 145-157 (1988).</li><li>Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+markers+for+identified+neuroblasts+and+ganglion+mother+cells+in+the+Drosophila+central+nervous+system.">Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system.</a> Development. 116, 855-863 (1992).</li><li>Lerit, D. A., Smyth, J. T., Rusan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+asymmetry+for+proper+fitness,+function,+and+fate.">Organelle asymmetry for proper fitness, function, and fate.</a> Chromosome research. 21, 271-286 (2013).</li><li>Bello, B. C., Izergina, N., Caussinus, E., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+neural+stem+cell+proliferation+by+intermediate+progenitor+cells+in+Drosophila+brain+development.">Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development.</a> Neural development. 3, 5 (2008).</li><li>Bowman, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+suppressors+Brat+and+Numb+regulate+transit-amplifying+neuroblast+lineages+in+Drosophila.">The tumor suppressors Brat and Numb regulate transit-amplifying neuroblast lineages in Drosophila.</a> Developmental Cell. 14, 535-546 (2008).</li><li>Boone, J. Q., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Drosophila+type+II+neuroblast+lineages+containing+transit+amplifying+ganglion+mother+cells.">Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells.</a> Dev Neurobiol. 68, 1185-1195 (2008).</li><li>Homem, C. C., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuroblasts:+a+model+for+stem+cell+biology.">Drosophila neuroblasts: a model for stem cell biology.</a> Development. 139, 4297-4310 (2012).</li><li>Savoian, M. S., Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+in+primary+cultures+of+Drosophila+melanogaster+larval+neuroblasts.">Mitosis in primary cultures of Drosophila melanogaster larval neuroblasts.</a> Journal of Cell Science. 115, 3061-3072 (2002).</li><li>Fleming, S. L., Rieder, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flattening+Drosophila+cells+for+high-resolution+light+microscopic+studies+of+mitosis+in+vitro.">Flattening Drosophila cells for high-resolution light microscopic studies of mitosis in vitro.</a> Cell Motil Cytoskeleton. 56, 141-146 (2003).</li><li>Buffin, E., Lefebvre, C., Huang, J., Gagou, M. E., Karess, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+Mad2+to+the+kinetochore+requires+the+Rod/Zw10+complex.">Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex.</a> Current biology. 15, 856-861 (2005).</li><li>Basto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flies+without+centrioles.">Flies without centrioles.</a> Cell. 125, 1375-1386 (2006).</li><li>Lucas, E. P., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+the+proper+connection+between+the+centrioles+and+the+pericentriolar+matrix+requires+Drosophila+centrosomin.">Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila centrosomin.</a> J. Cell Biol. 178, 725-732 (2007).</li><li>Basto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+amplification+can+initiate+tumorigenesis+in+flies.">Centrosome amplification can initiate tumorigenesis in flies.</a> Cell. 133, 1032-1042 (2008).</li><li>Conduit, P. T., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cnn+dynamics+drive+centrosome+size+asymmetry+to+ensure+daughter+centriole+retention+in+Drosophila+neuroblasts.">Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts.</a> Curr. Biol. 20, 2187-2192 (2010).</li><li>Forer, A., Pickett-Heaps, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochalasin+D+and+latrunculin+affect+chromosome+behaviour+during+meiosis+in+crane-fly+spermatocytes.">Cytochalasin D and latrunculin affect chromosome behaviour during meiosis in crane-fly spermatocytes.</a> Chromosome research. 6, 533-549 (1998).</li><li>Rebollo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+unequal+centrosomes+drive+spindle+orientation+in+asymmetrically+dividing+Drosophila+neural+stem+cells.">Functionally unequal centrosomes drive spindle orientation in asymmetrically dividing Drosophila neural stem cells.</a> Dev. Cell. 12, 467-474 (2007).</li><li>Januschke, J., Llamazares, S., Reina, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuroblasts+retain+the+daughter+centrosome.">Drosophila neuroblasts retain the daughter centrosome.</a> Nat. Commun. 2, 243 (2011).</li><li>Januschke, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrobin+controls+mother-daughter+centriole+asymmetry+in+Drosophila+neuroblasts.">Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts.</a> Nat Cell Biol. 15, 241-248 (2013).</li><li>Homem, C. C., Reichardt, I., Berger, C., Lendl, T., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Live+Cell+Imaging+and+Automated+4D+Analysis+of+Drosophila+Neuroblast+Lineages.">Long-Term Live Cell Imaging and Automated 4D Analysis of Drosophila Neuroblast Lineages.</a> PLoS One. 8, (2013).</li><li>Neumuller, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+self-renewal+in+Drosophila+neural+stem+cells+by+transgenic+RNAi.">Genome-wide analysis of self-renewal in Drosophila neural stem cells by transgenic RNAi.</a> Cell Stem Cell. 8, 580-593 (2011).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Siegrist, S. E., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+cues+orient+the+cell+division+axis+in+Drosophila+embryonic+neuroblasts.">Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts.</a> Development. 133, 529-536 (2006).</li><li>Broadus, J., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+cues,+intrinsic+cues+and+microfilaments+regulate+asymmetric+protein+localization+in+Drosophila+neuroblasts.">Extrinsic cues, intrinsic cues and microfilaments regulate asymmetric protein localization in Drosophila neuroblasts.</a> Curr. Biol. 7, 827-835 (1997).</li><li>Siller, K. H., Serr, M., Steward, R., Hays, T. S., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+brain+neuroblasts+reveals+a+role+for+Lis1/dynactin+in+spindle+assembly+and+mitotic+checkpoint+control.">Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control.</a> Molecular biology of the cell. 16, 5127-5140 (2005).</li><li>Britton, J. S., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+control+of+the+cell+cycle+in+Drosophila:+nutrition+activates+mitotic+and+endoreplicative+cells+by+distinct+mechanisms.">Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms.</a> Development. 125, 2149-2158 (1998).</li><li>Siller, K. H., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lis1/dynactin+regulates+metaphase+spindle+orientation+in+Drosophila+neuroblasts.">Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts.</a> Developmental Biology. 319, 1-9 (2008).</li><li>Cabernard, C., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical/basal+spindle+orientation+is+required+for+neuroblast+homeostasis+and+neuronal+differentiation+in+Drosophila.">Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila.</a> Dev. Cell. 17, 134-141 (2009).</li><li>Januschke, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interphase+microtubule+aster+is+a+determinant+of+asymmetric+division+orientation+in+Drosophila+neuroblasts.">The interphase microtubule aster is a determinant of asymmetric division orientation in Drosophila neuroblasts.</a> J. Cell Biol. 188, 693-706 (2010).</li><li>Cabernard, C., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+neuroblast+lineages+within+intact+larval+brains+in+Drosophila.">Live imaging of neuroblast lineages within intact larval brains in Drosophila.</a> Cold Spring Harb Protoc. 2013, 970-977 (2013).</li><li>Rusan, N. M., Akong, K., Peifer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putting+the+model+to+the+test:+are+APC+proteins+essential+for+neuronal+polarity,+axon+outgrowth,+and+axon+targeting.">Putting the model to the test: are APC proteins essential for neuronal polarity, axon outgrowth, and axon targeting.</a> J. Cell Biol. 183, 203-212 (2008).</li><li>Lerit, D. A., Rusan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLP+inhibits+the+activity+of+interphase+centrosomes+to+ensure+their+proper+segregation+in+stem+cells.">PLP inhibits the activity of interphase centrosomes to ensure their proper segregation in stem cells.</a> J. Cell Biol. 202, 1013-1022 (2013).</li><li>Morris, L. X., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+live+imaging+provides+new+insight+into+stem+cell+regulation+and+germline-soma+coordination+in+the+Drosophila+ovary.">Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary.</a> Development. 138, 2207-2215 (2011).</li><li>Prasad, M., Jang, A. C., Starz-Gaiano, M., Melani, M., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+culturing+Drosophila+melanogaster+stage+9+egg+chambers+for+live+imaging.">A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging.</a> Nat. Protoc. 2, 2467-2473 (2007).</li><li>Rusan, N. M., Peifer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+a+novel+centrosome+cycle+in+asymmetric+cell+division.">A role for a novel centrosome cycle in asymmetric cell division.</a> J. Cell Biol. 177, 13-20 (2007).</li><li>Martinez-Campos, M., Basto, R., Baker, J., Kernan, M., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+pericentrin-like+protein+is+essential+for+cilia/flagella+function,+but+appears+to+be+dispensable+for+mitosis.">The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis.</a> J. Cell Biol. 165, 673-683 (2004).</li><li>Dix, C. I., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Spd-2+recruits+PCM+to+the+sperm+centriole,+but+is+dispensable+for+centriole+duplication.">Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication.</a> Curr. Biol. 17, 1759-1764 (2007).</li><li>Moutinho-Santos, T., Sampaio, P., Amorim, I., Costa, M., Sunkel, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+localisation+of+the+mitotic+POLO+kinase+shows+a+highly+dynamic+association+with+the+mitotic+apparatus+during+early+embryogenesis+in+Drosophila.">In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila.</a> Biol. Cell. 91, 585-596 (1999).</li><li>Megraw, T. L., Kilaru, S., Turner, F. R., Kaufman, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centrosome+is+a+dynamic+structure+that+ejects+PCM+flares.">The centrosome is a dynamic structure that ejects PCM flares.</a> J. Cell Sci. 115, 4707-4718 (2002).</li></ol>

Live cell imaging is an invaluable approach used to study the mechanisms that regulate ACD of stem cells, the differentiation of cell lineages, and the morphogenesis of complex tissues. Although research groups have historically employed a variety of techniques to visualize NB ACD, these approaches can be generally grouped into two categories that primarily differ with respect to whether the brain tissue is left intact or dissociated.
Imaging dissociated NBs has its advantages. For one, dissoc...

Stem cells maintain a balance of differentiation and self-renewal to generate cellular diversity during early development and replace damaged cells in adult tissues. Regulation of this homeostasis prevents both the loss and over-expansion of the stem cell population, which can result in deleterious tissue degeneration or tumorigenesis.
Neuroblasts (NBs) are the widely studied neural stem cells of the Drosophila central nervous system (Figure 1A) that first form during embryonic stages1,2. NBs undergo repeated rounds of asymmetric cell division (ACD) to produce two unequally fated cells: a self-renewing stem cell and a differentiating cell. ACD is guided by centrosomes, the non-membranous organelles that act as the microtubule-organizing centers of most cells3. During mitosis, NB centrosomes organize and orient the bipolar mitotic spindle along the apical-basal polarity axis. Upon cleavage of the dividing NB, apical fate determinants that specify the stem cell fate, and basal fate determinants that specify differentiation, are segregated into the unequal daughter cells.
In the larval central brain, two types of NBs may be distinguished by their number, position, transcription factor expression, and cell lineage (Figure 1A)4-6. Type I NBs are the most abundant, and about 90 of them populate the anterior and posterior sides of each optic lobe of the brain7. These NBs express the transcription factor Asense (Ase), and they characteristically divide into one self-renewing NB and one smaller ganglion mother cell (GMC; Figure 1B). Each GMC undergoes a single terminal division to generate two neurons or glia (Figure 1B). In contrast, the eight Type II NBs that populate the posterior side of each optic lobe lack Ase expression5. They undergo ACD to produce one self-renewing NB and one intermediate neural progenitor (INP).
The INP, in turn, divides asymmetrically three to five times. Each of these divisions results in regeneration of the INP and the production of a single GMC4. Collectively, the specific NB identity and the temporal order of GMC birth gives rise to the astounding neuronal diversity of the adult central nervous system.
Understanding the cell biology that underlies NB ACD has been vastly improved through the use of live cell imaging techniques. Published protocols used by researchers to image live NBs vary widely. Overall, however, these methods may be grouped into two general categories distinguished by whether the larval brain is left intact or mechanically dissociated. Both techniques have distinct advantages and disadvantages depending on the researcher&#8217;s application.
Early reports revealing live NB cell divisions involve some degree of manual dissociation of the larval brain. These protocols detail smearing8 or teasing apart9 the brain in order to grow short-term primary cultures of the NBs on glass coverslips. To improve imaging, the round NBs are usually flattened on the coverslips with either glass slides8 or agarose pads9. Although flattened cells have improved optics, these techniques often lead to NB mitotic defects, including regression of the cleavage furrow and the inability to divide more than one time. Therefore, protocols that involve both manual dissociation and physical distortion of the larval brain tissue are generally only suited to very short-term (i.e., one cell cycle or less) applications. Likewise, semi-squashing or completely flattening intact brains between glass slides and coverslips limits the time one may spend imaging a given specimen to an approximately 30 min period10. Despite this limitation, this approach has been used successfully11-14.
Recent efforts to image live dissociated NBs limit physical distortion of the isolated cells. These techniques are particularly useful for applications where it is necessary to sustain cell cultures for multiple cell division cycles. For example, dispersed cells from manually dissociated brains may be partially embedded in a clot made from the mixture of fibrinogen and thrombin15 for long-term imaging16. Alternatively, explanted brains may be first chemically dissociated with the enzyme collagenase, next manually disrupted by repeated passage through a pipet tip, and subsequently plated on poly-L-lysine-coated glass bottom dishes17,18. Coating glass dishes with either fibrinogen or poly-L-lysine promotes the attachment and slight spreading of round cells, bringing them as close to the coverslip as possible without major physical distortion. In addition, these methods involve culturing the NBs in medium that may be readily exchanged for pharmacological perturbation experiments18. Overall, directly plating dispersed NBs improves imaging optics without sacrificing long-term imaging capabilities. Recently, a protocol describing the long-term culturing of dissociated NBs has been detailed19.
However, this approach is not without its limitations. There are several caveats to imaging live dissociated NBs. In a field of dispersed cells, for example, it is difficult to identify specific NB lineages that are spatially organized in the intact brain1. Likewise, distinguishing the Type I versus Type II NBs within dissociated tissue is also challenging without the expression of lineage tracers or subtype-specific transgenes, such as worniu-GAL4, asense-GAL8020. Although these transgenes are quite useful for some applications, they do limit researchers to those transgenes expressed under the control of the UAS enhancer element21 and require more complex genetic schemes. Studies that concern the spatial or temporal development of NBs, therefore, require in vivo imaging in the context of an intact tissue.
Moreover, studies of dissociated embryonic NBs indicate that the physical contact of adjacent cells is critical for the interphase localization of key polarity determinants22,23. These studies show completely isolated NBs no longer maintain the invariant mitotic spindle axis. Instead, these cells display a more randomized spindle axis and wide angles of separation between successive GMC buds, perhaps due to the loss of apical centrosome positioning22. Although the relationship between the larval NBs and their neighboring cells has not been extensively studied, it is worth considering that the larval stem cell microenvironment may impinge upon cell polarity or other behaviors. To maintain the physiological context of larval NBs, we image ACD from whole brains.
Given the inherent limitations associated with imaging dissociated NBs, our lab and others have established protocols to image successive rounds of NB ACD from whole larval brains. Techniques to image intact brains often replace the rigid surface of glass on one side of the culture chamber with a flexible membrane to prevent tissue distortion or damage and allow for gas exchange. Some methods involve mounting explanted brains in a mixture of insect culturing medium, serum, and ascorbic acid with the fat bodies isolated from ten larvae24. The isolated fat bodies are added because they secrete a mitogen known to stimulate NB cell division25. This protocol preserves the integrity of the tissue for over 3&#160;hr&#160;and is, therefore, amenable to long-term imaging24,26-28. Recently, a protocol describing the long-term imaging of intact larval brains in the presence of ascorbic acid, serum, and fat bodies has been detailed29. Importantly, because the tissue is neither exposed nor immobilized, this approach is less useful for applications where culturing medium is exchanged (e.g., drug studies, immunodepletion, etc.).
Our lab has simplified the whole mount preparation of live larval brains for long-term imaging30,31. The main advantage to our protocol over others is its simplicity: our observations indicate neither serum, ascorbic acid, insulin, nor fat bodies are necessary additives to image successive rounds of NB ACD. Although additives, such as insulin, have been useful for the prolonged imaging of stem cell divisions32 and collective cell migration in Drosophila ovaries33, they appear to be dispensable for NB ACD, as our imaging medium consists of a simple mixture of standard insect cell culturing medium supplemented with antibiotic-antimycotic to prevent contamination. Through the use of reusable gas-permeable or glass bottom culture dishes, we have been able to sustain several rounds of successive NB ACDs. The same explanted samples can readily be used for immunofluorescence and other procedures with fixed tissue. In this protocol, we detail the dissection of intact larval brains, as well as the preparation of brains for both live and fixed analysis.

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Lumox Tissue Culture Dish 50X12 mm</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:119px;">94.6077.410</td>
			<td style="width:167px;">A reusable culture dish with a gas-permeable and optically clear film base.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Schneider&#39;s S2 Medium</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:119px;">21720-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Gibco Antibiotic-Antimycotic (100x)</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:119px;">15240-062</td>
			<td>A supplement containing penicillin (10,000 U/mL), streptomycin (10,000 mg/mL), and amphotericin B (25 mg/mL) that is added to Schneider&#39;s S2 media to prevent bacterial and fungal contamination.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Glass coverslips, #1.5 22X22 mm</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">12-541-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Halocarbon oil 700</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8898</td>
			<td>A high viscosity inert oil that is clear and colorless.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">pair of nickel-plated pin holders, 17 cm long</td>
			<td style="width:185px;">Fine Science Tools</td>
			<td style="width:119px;">26018-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Minutien dissecting pins</td>
			<td style="width:185px;">Fine Science Tools</td>
			<td style="width:119px;">26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">pair of Dumont #5 Forceps</td>
			<td style="width:185px;">Fine Science Tools</td>
			<td style="width:119px;">11252-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Dissecting needle/probe with plastic handle</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">08-965-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Syringe filter, 0.22 mm pore size</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">09-719A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Syringe 5 mL&#160;</td>
			<td style="width:185px;">BD Biosciences</td>
			<td style="width:119px;">309646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">plastic transfer pipet</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">S30467-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:380px;">paraformaldehyde, 32% EM-grade</td>
			<td style="width:185px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714</td>
			<td>CAUTION: Formaldehyde is toxic; it should be handled in a fume hood. Follow MSDS guidelines for safe handling.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">DAPI (4&#39;, 6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:119px;">D1306</td>
			<td>A dye that labels DNA and is excited by UV light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Aqua/PolyMount; Polysciences, Inc.</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">18606-20</td>
			<td>A water-soluble mounting medium.</td>
		</tr>
	</tbody>
</table>

live imaging of drosophila larval neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live imaging has elucidated a number of mechanisms that regulate NB ACD. Prior to image acquisition, it is necessary to determine the optimal imaging conditions. It is necessary to balance the frame capture rate with the duration of live cell imaging. For fine cellular analysis, for example, increased temporal and optical resolution may be required. When visualizing a single NB cell cycle (Figure 4A), the rate at which images are captured may be increased without introducing photodamage. Typically, singl...

Watch this Scientific Journal Video about Live Imaging of Drosophila Larval Neuroblasts at JoVE.com

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

live-imaging-of-drosophila-larval-neuroblasts

Research

JoVE Journal

Neuroscience

14.6K visualizações.  National Institutes of Health.  Este protocolo detalha um método simplificado utilizado para conduzir imagem de células vivas, no contexto de um cérebro de larvas intacta. Abordagens imagens de células vivas são de valor inestimável para o estudo de células-tronco neurais divisões assimétricas, bem como outros processos neurogênicas e de desenvolvimento, de forma consistente descobrir mecanismos que anteriormente eram negligenciados. 