We thank Matthew O'Connell, Maryam Jahanshahi, and Andreas Jenny for assistance. We thank Istv&#225;n And&#243; for plasmatocyte-specific antibodies, Utpal Banerjee for dome-meso-EBFP2 flies, Julian Martinez-Agosto for antp&gt;GFP flies, and Michael O'Connor for ptth and ptth&gt;grim flies. These methods were developed with support by the Kimmel Foundation, the Leukemia &amp; Lymphoma Society, NIH/NCI R01CA140451, NSF 1257939, DOD/NFRP W81XWH-14-1-0059, and NIH/NCI T32CA078207.

1. Circulating Hemocyte Concentration
<ol>
	<li>To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2 - 6 hr.</li>
	<li>Collect larvae in dissecting dish wells filled with 1x phosphate buffered saline (PBS, Table 1).</li>
	<li>For each larva, place 10 &#181;l 1x PBS in a microcentrifuge tube on ice and 10 &#181;l 1x PBS on a clean dissecting pad. Place the dissecting pad on an illuminated stereomicroscope base.</li>
	<li>Dry an individual larva by placing it on a tissue wipe before transferring it to a PBS drop on the dissection pad.</li>
	<li>Using a pair of forceps, gently tear open and carefully invert the cuticle to release the hemolymph. 
	NOTE: Take care not to scrape or jab the cuticle as this could potentially release sessile hemocytes29.</li>
	<li>Using a pipet, collect the hemolymph from the dissecting pad. Avoid collecting larval debris such as the fat body.</li>
	<li>Add the hemolymph to a microcentrifuge tube and mix by pipetting up and down. 
	NOTE: Several samples can be collected at once and kept on ice for up to an hour.</li>
	<li>Optional: Mix Trypan blue with the hemolymph sample in equal parts to dye and exclude dead cells. Count cells within 3 min of adding Trypan blue because it is toxic to cells.</li>
	<li>Mix the sample by pipetting up and down before loading 10 &#181;l into a hemocytometer chamber.</li>
	<li>If using an automated cell counter, set appropriate parameters for cell size and circularity. For example, set a minimum cell size of 2 &#181;m, a maximum cell size of 22 &#181;m, and a circularity of 75 - 80% roundness to detect normal, round hemocytes34. Experiment with different parameters to detect larger and spindle-shaped hemocytes, such as lamellocytes. Alternatively, use a hemocytometer to manually count hemocytes.</li>
</ol>
2. Circulating Hemocyte Immunohistochemistry
<ol>
	<li>To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2 - 6 hr.</li>
	<li>Place one coverslip in each well of a 6- or 12-well plate. 
	NOTE: Square coverslips (22 mm) fit in 6-well plates and round coverslips (18 mm diameter) fit in 12-well plates.</li>
	<li>Collect larvae in dissecting dish wells filled with 1x PBS.</li>
	<li>Place 5 &#181;l 1x PBS in the center of each coverslip.</li>
	<li>Place the plate on an illuminated stereomicroscope base.</li>
	<li>Dry an individual larva on a tissue wipe and then place in the PBS on the coverslip.</li>
	<li>Using a pair of forceps, gently tear and carefully invert the cuticle. Before removing the larval carcass, use it to spread the hemolymph evenly. 
	NOTE: Take care not to scrape or jab the cuticle as this could potentially release sessile hemocytes29.</li>
	<li>Repeat for all larvae, collecting the hemolymph of one larva per coverslip. Allow hemocytes to adhere to the coverslips for 5 - 8 min at room temperature RT. Do not exceed 30 min before fixation.</li>
	<li>Fix hemocytes by adding 5 &#181;l of 7.5% formaldehyde (Table 1) to each coverslip for 15 min at room temperature.</li>
	<li>Wash coverslips three times with 1x PBS. Tip the plate and aspirate PBS after each wash. To prevent run-off of the permeabilization solution in the next step, use the aspirator to remove excess PBS from perimeter of the coverslips.</li>
	<li>Add 100 &#181;l permeabilization solution (Table 1) to each coverslip for 20 min at RT.</li>
	<li>Tip and gently tap the plate to aspirate the permeabilization solution.</li>
	<li>Dilute primary antibody with antibody solution (Table 1) according to provider&#39;s specifications. Add at least 500 &#181;l primary antibody solution to coverslips in 12-well plates. Add at least 1.5 ml in 6-well plates. Incubate at 4 &#176;C overnight.</li>
	<li>Remove the primary antibody solution and wash coverslips with 1x PBS for 10 min on an orbital shaker, three times.</li>
	<li>Dilute secondary antibody with antibody solution according to provider&#39;s specifications. Add at least 500 &#181;l secondary antibody solution to coverslips in 12-well plates. Add at least 1.5 ml for 6-well plates. Incubate for at least 2 hr at RT.</li>
	<li>Remove the secondary antibody solution and wash coverslips with 1x PBS for 10 min on an orbital shaker as in Step 2.10, three times.</li>
	<li>During the wash steps, clean glass microscope slides with 70% ethanol (Table 1) using tissue wipes. For each coverslip, place 5 &#181;l mounting buffer (Table 1) on the glass slide. 
	NOTE: Two coverslips fit on one slide.</li>
	<li>Aspirate the final PBS wash.</li>
	<li>Use forceps to carefully remove the coverslips from the plates and place, inverted, on top of the mounting buffer. 
	Note: Microscope slides can be stored at 4 &#176;C prior to imaging. Maximum storage time depends on the stability of the antibodies used. 6- and 12-well plates can be rinsed and reused.</li>
</ol>
3. In Vivo Crystal Cell Melanization
<ol>
	<li>To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2-6 hours.</li>
	<li>Before beginning, set a heating source at 60 &#176;C. 
	NOTE: A thermal cycler program of 60 &#176;C for 10 min (followed by a 25 &#176;C hold) works best, but a water bath or other heating source is sufficient as long as the heat is distributed consistently and evenly over each larva.</li>
	<li>Collect larvae in dissecting dish wells filled with 1x PBS (Table 1).</li>
	<li>One at a time, dry a larva on a tissue wipe and place at the bottom of a PCR tube. Place each larva in a separate PCR tube. (See Figure 3A.)</li>
	<li>For consistent results, ensure that larvae stay at the bottom of the PCR tubes by chilling larvae in the tubes at 4 &#176;C for 10 - 15 min and/or gently tapping the tubes prior to heating.</li>
	<li>Place the PCR tubes in the thermal cycler (or water bath). Heat at 60 &#176;C for 10 min.</li>
	<li>Carefully remove larvae from the PCR tubes into dissecting dish wells filled with fresh 1x PBS.</li>
	<li>Dry larvae on a tissue wipe and arrange on a flat surface for imaging under a stereomicroscope.</li>
	<li>Score images of larvae blindly by multiple individuals.</li>
</ol>
4. Larval Lymph Gland Immunohistochemistry
NOTE: The lymph gland is located approximately one-third length from the anterior end of a larva slightly below the brain on the dorsal side. (See arrow in Figure 3B.) The lymph gland flanks the dorsal vessel and is most easily dissected attached to the mouth hooks or to the brain. Wild-type, third instar lymph glands are very small structures; the primary lobes are approximately 100 - 200 &#181;m in length. (See Figure 4A.)
<ol>
	<li>To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2 - 6 hr.</li>
	<li>Lymph gland dissection
	<ol>
		<li>For each experimental condition, add 1 ml 1x PBS (Table 1) to one well of a 24-well plate.</li>
		<li>Add 1 drop of 0.1% PBST (Table 1) to each well using a disposable transfer pipet. 
		NOTE: Detergent, such as Tween 20, is added to lower the surface tension, allowing the tissue to sink to the bottom of the well.</li>
		<li>Place the plate flat on ice.</li>
		<li>Collect larvae in dissecting dish wells filled with 1x PBS.</li>
		<li>Place a clean dissecting pad on an illuminated stereomicroscope base. Use a disposable transfer pipet to place small drops of 0.01% PBST (Table 1) on the pad. Transfer one larva to a PBST drop for dissection.</li>
		<li>Hold the larva with one pair of forceps approximately one-quarter length from the posterior end, dorsal side up.</li>
		<li>Use another pair of forceps to grab the cuticle immediately anterior to the forceps that are holding the larva. Gently pull the cuticle toward the anterior end until the mouth hooks are exposed. 
		NOTE: The aim is to peel the cuticle without disturbing any internal structures.</li>
		<li>Release the cuticle and use both forceps to cut the larva in two. Remove the posterior end from the PBST drop. 
		NOTE: If the cuticle does not peel all the way to the mouth hooks, cut the larva in two and remove the posterior end. Use one pair of forceps to hold the edge of the cuticle, and use the other pair of forceps to push the mouth hooks through the opening. This will invert the cuticle and expose the mouth hooks. This can be used as an alternate dissection method as well.</li>
		<li>Use one pair of forceps to pin down the cuticle (either the ventral cuticle, the dorsal cuticle flap, or both) for stability.</li>
		<li>Use another pair of forceps to grab the exposed mouth hooks and gently pull them out. 
		NOTE: This will separate the cuticle from the internal structures. Ideally, the eye/antennal imaginal discs, brain, ring gland, and lymph gland flanking the dorsal vessel will remain attached to the mouth hooks.</li>
		<li>While still holding the mouth hooks, carefully remove unwanted structures such as the salivary glands, fat body, and intestine. 
		NOTE: If the brain separates from the mouth hooks, the lymph gland might still be attached to and collected with the brain. In this case, hold the ventral nerve cord instead of the mouth hooks.</li>
		<li>Using the mouth hooks or ventral nerve cord as a handle, transfer the dissected complex containing the lymph gland to the well on ice. Do not exceed 30 min before fixation.</li>
	</ol>
	</li>
	<li>Fixation and immunohistochemistry
	<ol>
		<li>Place the 24-well plate on the stereomicroscope base.</li>
		<li>Use a p200 pipette to carefully remove the PBST from the well. 
		NOTE: Empty, neighboring wells are useful to temporarily deposit waste.</li>
		<li>Gently add 200 &#181;l 3.7% formaldehyde (Table 1) down the side of the well and swirl the plate to ensure that the dissected tissues are completely submerged.</li>
		<li>Return the plate to ice for 30 min. If the lymph glands express fluorescent protein(s), keep the plate covered to prevent photobleaching.</li>
		<li>Use a p200 pipette to carefully remove the fixative.</li>
		<li>Wash by adding 200 &#181;l 1x PBS to the well and placing the plate on an orbital shaker for 5 min at room temperature. Use a p200 pipette to carefully remove the PBS. 
		NOTE: Fixed lymph glands can be left on ice in PBS until lymph glands for all experimental conditions are dissected and fixed.</li>
		<li>Repeat the wash steps two more times.</li>
		<li>Add 200 &#181;l permeabilization solution (Table 1) to the well. Set the plate on an orbital shaker for 45 min at RT. 
		NOTE: 45 min is the &#34;gold standard&#34; for lymph gland permeabilization but the authors have success after only 20 min.</li>
		<li>Remove the solution with a p200 pipette.</li>
		<li>Dilute primary antibody with antibody solution (Table 1) according to provider&#39;s specifications. Add 300 &#181;l primary antibody solution to the well and ensure that the dissected tissues are completely submerged. Incubate at 4 &#176;C overnight.</li>
		<li>Use a p200 pipette to remove the primary antibody.</li>
		<li>Wash by adding 200 &#181;l 1x PBS to the well and placing the plate on an orbital shaker for 10 min at room temperature. Use a p200 pipette to carefully remove the PBS.</li>
		<li>Repeat the wash steps two more times.</li>
		<li>Dilute secondary antibody with antibody solution according to provider&#39;s specifications. Add 300 &#181;l secondary antibody solution to the well and ensure that the dissected tissues are completely submerged. Incubate on an orbital shaker for at least 2 hr at RT.</li>
		<li>Use a p200 pipette to remove the secondary antibody and wash as described in Steps 4.3.12 &#38; 4.3.13. Do not remove the PBS after the final wash.</li>
	</ol>
	</li>
	<li>Lymph gland mounting
	<ol>
		<li>Clean glass microscope slides using 70% ethanol (Table 1) and tissue wipes.</li>
		<li>For each experimental condition, place one drop of mounting buffer (Table 1) on the glass slide. 
		NOTE: Two conditions fit on one slide. Mounting buffer volume depends on the number of lymph glands to be mounted. Use 2 &#181;l for approximately 18 - 20 lymph glands, 1 &#181;l for 10 - 12, and 0.5 &#181;l for 5 or less.</li>
		<li>Place the microscope slide on an illuminated stereomicroscope base.</li>
		<li>For up to two conditions at a time, transfer all of the lymph glands from the well to the mounting buffer with forceps, using the mouth hooks or ventral nerve cord as a handle.</li>
		<li>Space the dissected tissues evenly in a circular or rectangular shape, spreading the mounting buffer in the process.</li>
		<li>For each of the dissected tissues, individually, slide one tong of the forceps under the dorsal vessel and gently pull toward the periphery of the mounting buffer. 
		NOTE: This will draw the lymph gland out from the rest of the dissected tissues and flatten the lymph gland on the glass.</li>
		<li>Using one tong of the forceps and a sawing motion, cut the dorsal vessel between the lymph gland and the brain.</li>
		<li>Move the rest of the dissected tissues to the opposite side of the lymph gland, at the outermost edge of the buffer. 
		NOTE: The unwanted dissected tissues will eventually form a perimeter around the lymph glands and serve as a support upon which the coverslip will rest.</li>
		<li>Repeat until all of the lymph glands are separated from the dissected tissues, reserving one of the unwanted dissected tissues to place in the center.</li>
		<li>Take a coverslip between two fingers. Check that the coverslip is free of dust and fingerprints. If necessary, use a tissue wipe and 70% ethanol to clean the coverslip.</li>
		<li>Ensuring that the edge of the coverslip is parallel to the edge of the glass slide, carefully lower the coverslip over the mounting buffer. 
		NOTE: Microscope slides can be stored at 4 &#176;C prior to imaging. Maximum storage time depends on the stability of the antibodies used. 24-well plates can be rinsed and reused.</li>
	</ol>
	</li>
</ol>
5. Imaging
<ol>
	<li>Image fixed hemocytes and mounted lymph glands on an appropriate standard fluorescence or confocal microscope at 20X or higher magnification according to manufacturer&#39;s operating manual. Image whole larvae on a standard stereomicroscope at 2X magnification according to manufacturer&#39;s operating manual.</li>
	<li>Follow software provider&#39;s instructions for deconvolution, if desired.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Composition</td>
			<td>Storage</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td rowspan="5">1x PBS</td>
			<td>200 mg/L potassium chloride</td>
			<td rowspan="5">room temperature</td>
			<td rowspan="5"></td>
		</tr>
		<tr>
			<td>200 mg/L potassium phosphate monobasic</td>
		</tr>
		<tr>
			<td>8,000 mg/L sodium chloride</td>
		</tr>
		<tr>
			<td>1,150 mg/L sodium phosphate dibasic</td>
		</tr>
		<tr>
			<td>dH2O</td>
		</tr>
		<tr>
			<td>Fixative</td>
			<td>3.7% or 7.5% formaldehyde in 1x PBS</td>
			<td>room temperature in the dark</td>
			<td>Formaldehyde is toxic.&#160;</td>
		</tr>
		<tr>
			<td rowspan="3">Permeabilization solution/antibody diluent</td>
			<td>0.4% Triton</td>
			<td rowspan="3">4 &#176;C</td>
			<td rowspan="3">The standard formula uses 0.4% Triton but the authors use 0.1% Tween 20 with success. Use to dilute primary and secondary antibodies according to providers&#39; recommended concentrations.</td>
		</tr>
		<tr>
			<td>5% bovine serum albumin, normal goat serum, or normal donkey serum</td>
		</tr>
		<tr>
			<td>1x PBS</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>70% 200 proof ethanol in dH2O</td>
			<td>room temperature</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="4">Mounting buffer</td>
			<td>0.5% N-propyl gallate</td>
			<td rowspan="4">4 &#176;C in the dark</td>
			<td rowspan="4">N-propyl gallate is harmful. DAPI is a mutagen.</td>
		</tr>
		<tr>
			<td>80% glycerol</td>
		</tr>
		<tr>
			<td>Optional: 1 &#956;g/ml DAPI (4&#8217;,6-diamidino-2-phenylindole)</td>
		</tr>
		<tr>
			<td>1x PBS</td>
		</tr>
		<tr>
			<td>0.1% PBST</td>
			<td>0.1% Tween 20 in 1x PBS</td>
			<td>room temperature</td>
			<td></td>
		</tr>
		<tr>
			<td>0.01% PBST</td>
			<td>1:10 dilution of 0.1% PBST</td>
			<td>room temperature</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Solutions used in this Protocol.

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A., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+role+of+wingless+signaling+in+stem-like+hematopoietic+precursor+maintenance+in+Drosophila.">Dual role of wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila.</a> Dev Cell. 16 (5), 756-763 (2009).</li><li>Pennetier, D., 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=Size+control+of+the+Drosophila+hematopoietic+niche+by+bone+morphogenetic+protein+signaling+reveals+parallels+with+mammals.">Size control of the Drosophila hematopoietic niche by bone morphogenetic protein signaling reveals parallels with mammals.</a> Proc Natl Acad Sci U S A. 109 (9), 3389-3394 (2012).</li><li>Dragojlovic-Munther, M., Martinez-Agosto, 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=Extracellular+matrix-modulated+Heartless+signaling+in+Drosophila+blood+progenitors+regulates+their+differentiation+via+a+Ras/ETS/FOG+pathway+and+target+of+rapamycin+function.">Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function.</a> Dev Biol. 384 (2), 313-330 (2013).</li><li>Ferguson, G. B., Martinez-Agosto, 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=Yorkie+and+Scalloped+signaling+regulates+Notch-dependent+lineage+specification+during+Drosophila+hematopoiesis.">Yorkie and Scalloped signaling regulates Notch-dependent lineage specification during Drosophila hematopoiesis.</a> Curr Biol. 24 (22), 2665-2672 (2014).</li><li>Milton, C. 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=The+Hippo+pathway+regulates+hematopoiesis+in+Drosophila+melanogaster.">The Hippo pathway regulates hematopoiesis in Drosophila melanogaster.</a> Curr Biol. 24 (22), 2673-2680 (2014).</li><li>Evans, C. J., Liu, T., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+hematopoiesis:+Markers+and+methods+for+molecular+genetic+analysis.">Drosophila hematopoiesis: Markers and methods for molecular genetic analysis.</a> Methods. 68 (1), 242-251 (2014).</li><li>Neyen, C., Bretscher, A. J., Binggeli, O., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+study+Drosophila+immunity.">Methods to study Drosophila immunity.</a> Methods. 68 (1), 116-128 (2014).</li><li>Small, C., Paddibhatla, I., Rajwani, R., Govind, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+parasitic+wasps+of+Drosophila+and+the+antiparasite+immune+response.">An introduction to parasitic wasps of Drosophila and the antiparasite immune response.</a> J Vis Exp. (63), e3347 (2012).</li><li>Petraki, S., Alexander, B., Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+Blood+Cell+Populations+of+the+Drosophila+melanogaster+Larva.">Assaying Blood Cell Populations of the Drosophila melanogaster Larva.</a> J Vis Exp. (105), (2015).</li><li>Rizki, M. T. M., Rizki, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+significance+of+the+crystal+cells+in+the+larva+of+Drosophila+mekmogaster.">Functional significance of the crystal cells in the larva of Drosophila mekmogaster.</a> Journal of Biophysical and Biochemical Cytology. 5, 235-240 (1959).</li><li>Kurucz, 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=Definition+of+Drosophila+hemocyte+subsets+by+cell-type+specific+antigens.">Definition of Drosophila hemocyte subsets by cell-type specific antigens.</a> Acta Biol Hung. 58, 95-111 (2007).</li><li>Jung, S. H., Evans, C. J., Uemura, C., Banerjee, U. <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+lymph+gland+as+a+developmental+model+of+hematopoiesis.">The Drosophila lymph gland as a developmental model of hematopoiesis.</a> Development. 132 (11), 2521-2533 (2005).</li><li>Krzemien, J., Crozatier, M., Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+the+Drosophila+larval+hematopoietic+organ,+hemocyte+homeostasis+and+the+dedicated+cellular+immune+response+to+parasitism.">Ontogeny of the Drosophila larval hematopoietic organ, hemocyte homeostasis and the dedicated cellular immune response to parasitism.</a> Int J Dev Biol. 54 (6-7), 1117-1125 (2010).</li><li>Rizki, T. M., Rizki, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+Larval+Hemocytes+of+Drosophila-Melanogaster.">Properties of the Larval Hemocytes of Drosophila-Melanogaster.</a> Experientia. 36 (10), 1223-1226 (1980).</li><li>Lanot, R., Zachary, D., Holder, F., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postembryonic+hematopoiesis+in+Drosophila.">Postembryonic hematopoiesis in Drosophila.</a> Dev Biol. 230 (2), 243-257 (2001).</li><li>McBrayer, Z., 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=Prothoracicotropic+hormone+regulates+developmental+timing+and+body+size+in+Drosophila.">Prothoracicotropic hormone regulates developmental timing and body size in Drosophila.</a> Dev Cell. 13 (6), 857-871 (2007).</li><li>Reimels, T. A., Pfleger, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Rabex-5+restricts+Notch+activity+in+hematopoietic+cells+and+maintains+hematopoietic+homeostasis.">Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis.</a> J Cell Sci. 128 (24), 4512-4525 (2015).</li></ol>

The authors declare that they have no competing or financial interests.

Upon genetic or environmental alteration, the four methods described here can be used individually or in conjunction to analyze distinct processes during hematopoiesis such as signaling, survival, proliferation, and differentiation. Drosophila hematopoiesis is a dynamic process; the number of hemocytes per animal increases35 and the structure and gene expression of the lymph gland changes32 during development. Prior to performing these assays, therefore, it is critical to restrict egg colle...

The complex mechanisms regulating the transcription factors and signaling pathways that coordinate the development of the hematopoietic system and that malfunction in hematological diseases remain poorly understood. These transcription factors and signaling pathways, as well as their regulation, are highly conserved between Drosophila and mammalian hematopoiesis1-5. Thus the Drosophila hematopoietic system represents an excellent genetic model to define the molecular mechanisms controlling hematopoiesis and underlying hematological diseases.
Similar to mammals, Drosophila generate blood cells, called hemocytes, in spatially and temporally distinct phases of hematopoiesis. Traditionally, Drosophila hematopoiesis was thought to be restricted to phases in the embryonic mesoderm and in the larval lymph gland. Recent studies provide evidence that hematopoiesis also occurs in larval sessile clusters and in the adult abdomen6-8. All hematopoietic phases produce two types of mature hemocytes: plasmatocytes and crystal cells. Plasmatocytes are macrophage-like cells involved in phagocytosis, innate immunity, and wound healing. Crystal cells contain pro-phenoloxidases required for melanization, a reaction used in insect immune responses and wound healing. Larval hematopoiesis can generate a third mature hemocyte type, called a lamellocyte, in response to certain immune challenges such as parasitoid wasp infection9,10. Lamellocytes are large, adherent cells that function, in conjunction with plasmatocytes and crystal cells, to encapsulate and neutralize wasp eggs laid in Drosophila larvae. In the absence of parasitization, lamellocytes are not found in wild-type larvae. Melanotic masses resemble melanized, encapsulated wasp eggs; many mutant Drosophila strains develop melanotic masses in the absence of parasitization. The presence of lamellocytes and/or melanotic masses can be indicative of hematopoietic abnormalities. In fact, the melanotic mass phenotype has been used to identify genes and pathways involved in hematopoiesis11-14.
The larval hematopoietic system is the most extensively studied to date. It is comprised of hemocytes circulating in the hemolymph, sessile hemocyte clusters patterned under the cuticle, and hemocytes residing in the lymph gland. The lymph gland is a series of bilateral lobes attached to the dorsal vessel. Each primary lobe of the lymph gland is divided into three main zones. The outermost zone is known as the cortical zone and contains maturing hemocytes. The innermost zone is called the medullary zone and is comprised of quiescent hemocyte precursors. The third zone, the posterior signaling center, is a small group of cells at the base of the lymph gland that act as a stem cell-like niche. Early work established critical functions for Notch15-18, Hedgehog19,20, JAK-STAT18, and Wingless21 activity to regulate larval lymph gland development. More recent studies have demonstrated that BMP22, FGF-Ras23, and Hippo24,25 signaling also function within the larval lymph gland.
Four larval hematopoietic assays outlined here describe 1) measuring circulating hemocyte concentration, defined as number of cells per unit volume, 2) isolating and fixing circulating hemocytes for immunohistochemistry, 3) visualizing crystal cells in vivo, and 4) dissecting, fixing, and mounting lymph glands for immunohistochemistry. These assays can be used as hematopoietic readouts to assess the functions and regulations of signaling pathways in the larval hematopoietic system. While these methods have been used previously in the field, visual documentation of these assays has begun only recently8,26-30. Several publications cited here are helpful resources describing similar methods and hematopoietic markers26,31-33. Additionally, Trol and Viking are useful markers of the lymph gland basement membrane.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">PBS tablets</td>
			<td style="width:176px;">MP Biomedicals</td>
			<td style="width:163px;">2810305</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">dissecting dish</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">7220-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">microcentrifuge tube</td>
			<td style="width:176px;">Denville</td>
			<td style="width:163px;">C2170</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:229px;">silicone dissecting pad, made from Sylgard 184 kit</td>
			<td style="width:176px;">Krayden (distributed through Fisher)</td>
			<td style="width:163px;">NC9644388 (Fisher catalog number)</td>
			<td style="width:236px;">Made in petri dish by mixing components of Sylgard elastomer kit according to manufacturer instructions.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">stereomicroscope</td>
			<td style="width:176px;">Morrell Instruments (Nikon distributor)</td>
			<td>mna42000, mma36300</td>
			<td>Nikon models SMZ1000 and SMZ645</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">tissue wipe</td>
			<td style="width:176px;">VWR</td>
			<td style="width:163px;">82003-820</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">forceps</td>
			<td style="width:176px;">Electron Microscopy Sciences</td>
			<td style="width:163px;">72700-DZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">p200 pipette</td>
			<td style="width:176px;">Eppendorf</td>
			<td>3120000054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Countess Automated Cell Counter</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:163px;">C10227</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Countess cell counting chamber slides</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:163px;">C10283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">hemocytometer</td>
			<td style="width:176px;">Hausser Scientific</td>
			<td style="width:163px;">3200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">trypan blue stain</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:163px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">formaldehyde</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">BP531-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Triton</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">BP151-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Tween 20</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">BP337-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">bovine serum albumin</td>
			<td style="width:176px;">Rocky Mountain Biologicals</td>
			<td style="width:163px;">BSA-BSH-01K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">normal goat serum</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:163px;">G9023-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">normal donkey serum</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:163px;">D9663-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">200 proof ethanol</td>
			<td style="width:176px;">VWR</td>
			<td style="width:163px;">V1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">N-propyl gallate</td>
			<td style="width:176px;">MP Biomedicals</td>
			<td style="width:163px;">102747</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">glycerol</td>
			<td style="width:176px;">VWR</td>
			<td style="width:163px;">EM-4750</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">DAPI (4&#8217;,6-diamidino-2-phenylindole)</td>
			<td style="width:176px;">Fisher</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">6-well plate</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">12-well plate</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">351143</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">microscope cover glass, 22 mm square</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">12-544-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">microscope cover glass, 18mm circular</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">12-545-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">glass microscope slides</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">22-034-980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">thermal cycler</td>
			<td style="width:176px;">Eppendorf</td>
			<td>E950010037</td>
			<td>Mastercycler EP Gradient S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">PCR tubes</td>
			<td style="width:176px;">USA Scientific</td>
			<td style="width:163px;">1402-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">24-well plate</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">351147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">disposable transfer pipet</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">13-711-9AM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">fluorescence microscope</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:163px;"></td>
			<td>Axio Imager.Z1</td>
		</tr>
	</tbody>
</table>

methods to examine the lymph gland and hemocytes in drosophila larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Circulating Hemocyte Concentration
Hemocyte numbers increase throughout larval development35. To illustrate that this method detects differences in hemocyte numbers and concentration, regardless of the biological cause, we measured hemocyte concentrations of delayed and non-delayed larvae. Loss of prothoracicotropic hormone (ptth) by genetic ablation of ptth-producing neurons (ptth&#62;grim) produces a delay in larval de...

Watch this Scientific Journal Video about Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae at JoVE.com

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

methods-to-examine-the-lymph-gland-and-hemocytes-in-drosophila-larvae

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JoVE Journal

Developmental Biology

12.7K Views.  The Icahn School of Medicine at Mount Sinai. Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Video: Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...