Name: organelle transport in cultured drosophila cells: s2 cell line and primary neurons.
Uploaded: 2013-11-20T18:00:00
Description: Watch this Scientific Journal Video about Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons. at JoVE.com

We thank the members of the Gelfand lab who contributed to the development of these protocols. Research reported in this publication was supported by the National Institute of General Medical Science of the National Institutes of Health under award number R01GM052111 to VIG and by Basque Government Department of Education, Universities and Research under award number BFI-2011-295 to UdC.

1. Drosophila S2 cells
<ol>
	<li>Preparation of ConA coated coverslips
	<ol>
		<li>Place coverslips in a ceramic rack and wash them by chromic acid immersion for 1 hr. [CAUTION: chromic acid is corrosive and can cause irritation of eyes, nose, throat, and skin].</li>
		<li>Rinse coverslips thoroughly with constantly running dH2O&#160;for 30 min until the acid is completely washed out.</li>
		<li>Let coverslips air dry and coat them with ConA (0.5 mg/ml solution in dH2O) f...

<ol>
	<li>Schneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines+derived+from+late+embryonic+stages+of+Drosophila+melanogaster.">Cell lines derived from late embryonic stages of Drosophila melanogaster.</a> J. Embryol. Exp. Morphol. 27, 353-365 (1972).</li><li>Rogers, S. L., Rogers, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Drosophila+S2+cells+and+their+use+for+RNAi-mediated+loss-of-function+studies+and+immunofluorescence.">Culture of Drosophila S2 cells and their use for RNAi-mediated loss-of-function studies and immunofluorescence.</a> 3, 606-611 (2008).</li><li>Buster, D. W., Nye, J., Klebba, J. E., Rogers, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Drosophila+S2+cells+for+light+microscopy.">Preparation of Drosophila S2 cells for light microscopy.</a> J. Vis. Exp. (40), e1982 (2010).</li><li>Ling, S. C., Fahrner, P. S., Greenough, W. T., Gelfand, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+Drosophila+fragile+X+mental+retardation+protein-containing+ribonucleoprotein+granules+by+kinesin-1+and+cytoplasmic+dynein.">Transport of Drosophila fragile X mental retardation protein-containing ribonucleoprotein granules by kinesin-1 and cytoplasmic dynein.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 17428-17433 (2004).</li><li>Kural, C., 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=Kinesin+and+dynein+move+a+peroxisome+in+vivo:+a+tug-of-war+or+coordinated+movement.">Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement.</a> Science. 308, 1469-1472 (2005).</li><li>Kim, H., 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=Microtubule+binding+by+dynactin+is+required+for+microtubule+organization+but+not+cargo+transport.">Microtubule binding by dynactin is required for microtubule organization but not cargo transport.</a> J. Cell. Biol. 176, 641-651 (2007).</li><li>Ally, S., Larson, A. G., Barlan, K., Rice, S. E., Gelfand, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite-polarity+motors+activate+one+another+to+trigger+cargo+transport+in+live+cells.">Opposite-polarity motors activate one another to trigger cargo transport in live cells.</a> J. Cell. Biol. 187, 1071-1082 (2009).</li><li>Bensenor, L. B., Barlan, K., Rice, S. E., Fehon, R. G., Gelfand, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-mediated+transport+of+the+tumor-suppressor+protein+Merlin+and+its+mutants.">Microtubule-mediated transport of the tumor-suppressor protein Merlin and its mutants.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 7311-7316 (2010).</li><li>Jolly, A. L., 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=Kinesin-1+heavy+chain+mediates+microtubule+sliding+to+drive+changes+in+cell+shape.">Kinesin-1 heavy chain mediates microtubule sliding to drive changes in cell shape.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 12151-12156 (2010).</li><li>Barlan, K., Lu, W., Gelfand, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microtubule-binding+protein+ensconsin+is+an+essential+cofactor+of+Kinesin-1.">The microtubule-binding protein ensconsin is an essential cofactor of Kinesin-1.</a> Curr. Biol. 23, 317-322 (2013).</li><li>Lu, W., Fox, P., Lakonishok, M., Davidson, M. W., Gelfand, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+Neurite+Outgrowth+in+Drosophila+Neurons+Is+Driven+by+Kinesin-Powered+Microtubule+Sliding.">Initial Neurite Outgrowth in Drosophila Neurons Is Driven by Kinesin-Powered Microtubule Sliding.</a> Curr. Biol. 23, 1018-1023 (2013).</li><li>Pathak, D., Sepp, K. J., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+myosin+activity+opposes+microtubule-based+axonal+transport+of+mitochondria.">Evidence that myosin activity opposes microtubule-based axonal transport of mitochondria.</a> J. Neurosci. 30, 8984-8992 (2010).</li><li>Clark, I. 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=Drosophila+pink1+is+required+for+mitochondrial+function+and+interacts+genetically+with+parkin.">Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin.</a> Nature. 441, 1162-1166 (2006).</li><li>Gunawardena, S., 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=Disruption+of+axonal+transport+by+loss+of+huntingtin+or+expression+of+pathogenic+polyQ+proteins+in+Drosophila.">Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila.</a> Neuron. 40, 25-40 (2003).</li><li>Koo, E. H., 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=Precursor+of+amyloid+protein+in+Alzheimer+disease+undergoes+fast+anterograde+axonal+transport.">Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport.</a> Proc. Natl. Acad. Sci. U.S.A. 87, 1561-1565 (1990).</li><li>Stokin, G. B., Goldstein, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+and+Alzheimer's+disease.">Axonal transport and Alzheimer's disease.</a> Annu. Rev. Biochem. 75, 607-627 (2006).</li><li>Bilsland, L. G., 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=Deficits+in+axonal+transport+precede+ALS+symptoms+in+vivo.">Deficits in axonal transport precede ALS symptoms in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 20523-20528 (2010).</li><li>Lloyd, T. 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=The+p150(Glued)+CAP-Gly+domain+regulates+initiation+of+retrograde+transport+at+synaptic+termini.">The p150(Glued) CAP-Gly domain regulates initiation of retrograde transport at synaptic termini.</a> Neuron. 74, 344-360 (2012).</li><li>Moughamian, A. J., Holzbaur, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynactin+is+required+for+transport+initiation+from+the+distal+axon.">Dynactin is required for transport initiation from the distal axon.</a> Neuron. 74, 331-343 (2012).</li><li>Lorenzo, D. N., 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=Spectrin+mutations+that+cause+spinocerebellar+ataxia+type+5+impair+axonal+transport+and+induce+neurodegeneration+in+Drosophila.">Spectrin mutations that cause spinocerebellar ataxia type 5 impair axonal transport and induce neurodegeneration in Drosophila.</a> J. Cell. Biol. 189, 143-158 (2010).</li><li>Robinow, S., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+locus+elav+of+Drosophila+melanogaster+is+expressed+in+neurons+at+all+developmental+stages.">The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages.</a> Dev. Biol. 126, 294-303 (1988).</li><li>Pilling, A. D., Horiuchi, D., Lively, C. M., Saxton, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin-1+and+Dynein+are+the+primary+motors+for+fast+transport+of+mitochondria+in+Drosophila+motor+axons.">Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons.</a> Mol. Biol. Cell. 17, 2057-2068 (2006).</li><li>Furst, A., Mahowald, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+primary+embryonic+neuroblasts+in+purified+neural+cell+cultures+from+Drosophila.">Differentiation of primary embryonic neuroblasts in purified neural cell cultures from Drosophila.</a> Dev. Biol. 109, 184-192 (1985).</li><li>Salvaterra, P. M., Bournias-Vardiabasis, N., Nair, T., Hou, G., Lieu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+neuronal+differentiation+of+Drosophila+embryo+cells.">In vitro neuronal differentiation of Drosophila embryo cells.</a> J. Neurosci. 7, 10-22 (1987).</li><li>Wiemer, E. A., Wenzel, T., Deerinck, T. J., Ellisman, M. H., Subramani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+peroxisomal+compartment+in+living+mammalian+cells:+dynamic+behavior+and+association+with+microtubules.">Visualization of the peroxisomal compartment in living mammalian cells: dynamic behavior and association with microtubules.</a> J. Cell. Biol. 136, 71-80 (1997).</li><li>Rizzuto, 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=A+gene+specifying+subunit+VIII+of+human+cytochrome+c+oxidase+is+localized+to+chromosome+11+and+is+expressed+in+both+muscle+and+non-muscle+tissues.">A gene specifying subunit VIII of human cytochrome c oxidase is localized to chromosome 11 and is expressed in both muscle and non-muscle tissues.</a> J. Biol. Chem. 264, 10595-10600 (1989).</li><li>Poot, M., 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=Analysis+of+mitochondrial+morphology+and+function+with+novel+fixable+fluorescent+stains.">Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.</a> J. Histochem. Cytochem. 44, 1363-1372 (1996).</li><li>Haller, T., Dietl, P., Deetjen, P., Volkl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lysosomal+compartment+as+intracellular+calcium+store+in+MDCK+cells:+a+possible+involvement+in+InsP3-mediated+Ca2++release.">The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in InsP3-mediated Ca2+ release.</a> Cell Calcium. 19, 157-165 (1996).</li></ol>

Here we present detailed protocols for studying microtubule-dependent cargo transport in Drosophila S2 cells and primary neuron culture. Both S2 processes and neurites are structures filled by bundled microtubules arrays that serve as tracks for organelle transport.
Two strategies are typically used to label organelles: transfection of vectors encoding a fluorescent protein with an organelle-targeting sequence or staining with fluorescent dyes added directly to cultures. Common exampl...

Drosophila S2 cells have gained increasing popularity for studying many cellular processes. These cells were originally obtained from trypsinized late stage embryos of OregonR Drosophila melanogaster and maintain macrophage-like features1. Drosophila S2 cells offer many advantages over other cell lines. They are cultured at 25 &#176;C without CO2, and can be imaged for many hours at room temperature without the need of heating or gas exchange. More importantly, Drosophila S2 cells are very sensitive to RNAi treatment allowing high efficiency knockdown of proteins of interest. S2 cells are highly transfectable and stable cell lines can be selected in less than four weeks to allow stable ectopic expression of proteins of interest. S2 cells normally grow either loosely attached but not spread on a flask or in suspension. They can be induced to spread on coverslips precoated with a plant lectin concanavalin A (ConA). The periphery of the spread cells is especially thin and therefore is ideal for live cell microscopy. Detailed protocols for S2 cells culture, RNAi treatment, live light imaging and immunostaining can be found in the literature2,3. Our lab has adopted S2 cells as a model system to study microtubule-based cargo transport. Drosophila S2 cells treated with any drug that depolymerizes or severs F-actin, such as cytochalasin D (CytoD), grow long processes filled with uniformly polarized microtubules4-10, which makes it an ideal system to study cargo movement along microtubule arrays. Different parameters of transport in these processes (i.e. trajectories lengths, velocities, directionality etc.) can be easily measured using automated particle-tracking software, DiaTrack (Semasopht: http://www.microscopy.info/Organization/Details/4423; Dr. Pascal Vallotton: http://www.csiro.au/en/Organisation-Structure/Divisions/Mathematics-Informatics-and-Statistics/PascalVallotton.aspx#a4). These properties make S2 cells an appealing system for studying cargo transport along microtubules in tissue culture cells.
Pilot experiments in S2 cells can be extended to a physiologically important model of cargo transport studies in Drosophila primary neurons. Similar to S2 cells, neurons cultured from dissociated embryos are permeable to small molecules such as dyes and inhibitors, and high-resolution live imaging of organelle transport can be performed in neurons at room temperature. Using Drosophila with different genetic backgrounds, we can examine gene function in primary neurons by knockout (genetic loss-of-function mutations), knockdown (dsRNA injection or expression) or ectopic expression (transgenic flies). Specifically, fragmentation of actin filaments using CytoD treatment does not prevent neurite outgrowth; instead they grow faster, and thus we can study microtubule-dependent organelle transport in CytoD-treated neurons without the influence of actin filaments11. Therefore, primary neuronal cultures combine the advantages of tissue culture cells and fly genetics, which makes it a great system to study cargo transport in a physiological relevant system10,12. Since several familial neurodegenerative diseases could be caused by or associated with cargo transport defects (e.g.&#160;Parkinson&#39;s disease13, Huntington&#39;s disease14, Alzheimer&#39;s Disease15,16, Amyotrophic Lateral Sclerosis/ALS17, HMN7B and Perry syndrome18,19, and Spinocerebellar ataxia type 5/SCA520), primary neuronal cultures can be useful in analysis of the effects of specific mutations causing these diseases on microtubule-dependent transport.
Finally, important properties of microtubule-dependent transport are well conserved between mammals and flies, but Drosophila genetic and cell culture is less expensive and time-consuming, justifying the use of cultured Drosophila cells for studying essential aspects of organelle transport in normal and pathological conditions.

<table border="0" cellpadding="0" cellspacing="0" width="435">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Powder Insect cell medium (Schneider&#8217;s)</td>
			<td style="width:133px;">Sigma&#160;</td>
			<td style="width:179px;">S9895</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Insect &#8211;Xpress medium</td>
			<td style="width:133px;">Fisher</td>
			<td style="width:179px;">BW12730Q</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Insulin</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">I6634</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Penicillin</td>
			<td style="width:133px;">Research Products International</td>
			<td style="width:179px;">P93000</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Streptomycin</td>
			<td style="width:133px;">Research Products International</td>
			<td style="width:179px;">S62000</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Tetracycline hydrochloride</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">T7660-5G</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Fetal bovine serum</td>
			<td style="width:133px;">Atlanta Biologicals</td>
			<td style="width:179px;">S11150</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Concanavalin A</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">C2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Cytochalasin D</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">IC15077105</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">LysoTracker Red DND-99</td>
			<td style="width:133px;">Molecular Probes</td>
			<td style="width:179px;">L7528</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">100 &#181;m Cell strainer</td>
			<td style="width:133px;">Fisher Scientific</td>
			<td style="width:179px;">22363549</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">25 mm Circle cover glass</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">48380 080</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Drosophila Vials</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">89092-730</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:124px;">Disposable pellet pestles with matching microtubes</td>
			<td style="width:133px;">Kimble Chase</td>
			<td style="width:179px;">749520-0000</td>
		</tr>
	</tbody>
</table>

organelle transport in cultured drosophila cells: s2 cell line and primary neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Drosophila S2 cells attached on the ConA-coated coverslip spread into a flat round &#8220;pancake&#8221; shape (Figures 1A and 1C). However, when S2 cells plated on coverslips in the presence of CytoD and allowed to spread for 2-3 hr, they form long thin processes filled by parallel microtubules (Figures 1B and 1D). These microtubules have uniform polarity with plus-ends out7. Time-lapse fluorescence imaging of Drosophila S2 ...

Watch this Scientific Journal Video about Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons. at JoVE.com

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

organelle-transport-cultured-drosophila-cells-s2-cell-line-primary

Research

JoVE Journal

Biology

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...