We thank Dr. Guy and Dr. Kim Caldwell for the use of their NanoDrop spectrophotometer during the development of this assay&#160;and J. Gavin Daigle for his critical comments and suggestions. The development of this method was funded by a grant from the National Institutes of Health (NS-078728) to J.M.O.

1. Paraquat Exposure
<ol>
	<li>Maintain the cultures of the test flies on standard Drosophila culture medium at 25 &#176;C under equivalent culture densities consisting of approximately 35 females and 15 males.</li>
	<li>To prepare feeding chambers, place a small amount of absorbent cotton at the bottom of an empty vial with filter paper directly above the cotton.</li>
	<li>Anesthetize adult male flies 3-5 days posteclosion using cold coma or CO2. For the experiments described here, use males of genotype y w1118.
	<ol>
		<li>For cold coma separation, tap down feeding vials containing flies and then quickly remove stopper and transfer flies directly to a clean, empty vial, making sure to label properly.</li>
		<li>Once flies are successfully transferred to empty food vials, tap down vials then immediately place in ice bucket for 5 min or until flies are knocked out. 
		Note: Do not completely submerge vial in ice. This can cause water or moisture to get into the vial and drown the flies. As another precaution, use fresh ice rather than melted ice in ice buckets. 
		Note: Pay close attention to the number of flies needed for each assay and, when possible, add additional flies to each group as a precaution in the event of human error, e.g. flies escaping during feeding or transferring. The nitric oxide detection assay, for example, requires 20 fly brains per sample set, so account for dissection skill-level and efficiency when feeding (i.e. novices should set up more than 20 flies in case of transferring or dissection errors).</li>
	</ol>
	</li>
	<li>Once flies are anesthetized, transfer each group into corresponding feeding vial as prepared above.</li>
	<li>After the flies are fully awake in feeding vials, starve them for 1.5 hr and meanwhile, prepare solutions described below:
	<ol>
		<li>Prepare a stock solution of 5% sucrose (control solution) by adding 5 g of sucrose into 100 ml of double-distilled water. Stir until completely dissolved and store at 4 &#176;C. 
		Note: Use 5% sucrose stock solution as the solvent for all subsequent feeding solutions.</li>
		<li>Prepare stock 10 mM paraquat stock solution using 5% sucrose as diluent and stored at 4 &#176;C. Prepare all other concentrations using serial dilutions as needed. 
		CAUTION: PARAQUAT IS EXTREMELY TOXIC. REFER TO MSDS FOR SAFETY GUIDELINES AND PROPER HANDLING/DISPOSAL.</li>
		<li>Prepare 10 mM stock solutions of N-Nitro-L-arginine methyl ester hydrochloride (L-NAME), N-nitro-D-arginine methyl ester hydrochloride (D-NAME) using 5% sucrose as diluent and store at 4 &#176;C. For coincubations of L-NAME or D-NAME with paraquat administer each compound at a 1:1 ratio with paraquat concentration. Prepare all other concentrations using serial dilutions as needed. 
		Note: Filter all solutions directly after preparation. Paraquat, D-NAME, and L-NAME stocks should be used within one week of preparation to ensure experimental consistency.</li>
		<li>Pipette 150 &#956;l per day (or less depending on feeding duration) of the feeding solution onto the filter paper, being careful not to drown flies or let any escape.</li>
	</ol>
	</li>
</ol>
2. Survival/Mortality Assay
<ol>
	<li>Set up feeding chambers as previously described. 
	Note: Use at least 3 layers of filter paper per vial to prevent desiccation.</li>
	<li>Place 10 male flies 3-5 days posteclosion in each vial and feed as directed above, being cautious to not let the filter paper dry out overnight. 
	Note: Maintain vials at room temperature, away from direct sunlight to avoid accelerating evaporation of solutions.</li>
	<li>Beginning the following day (designated &#34;Day 1&#34;), record number of flies dead per vial twice a day for 10 consecutive days. 
	Note: Regulate both scoring and feeding to specific hours each day to decrease variation and provide higher consistency.</li>
	<li>Calculate average survival using at least three independent replications of all groups.</li>
</ol>
3. Dissecting Adult Fly Brains and Incubation
<ol>
	<li>Prior to dissections, pipette 50 &#956;l of Grace&#39;s Insect Medium into the appropriate number of wells (one well for each sample set + one for the &#34;blank&#34;/control) in a 96-well microtiter plate and label suitably. Cover with plate lid until use. 
	Note: Alternatively, sterile centrifuge tubes may also be used if preferred.</li>
	<li>Anesthetize flies of one sample set using cold coma as described in steps 1.3.1-1.3.2.</li>
	<li>Once flies are fully anesthetized, place several flies on a microscope slide 
	Note: Begin with a small subset of flies if not experienced with dissections.</li>
	<li>Decapitate the fly heads under a dissection microscope using forceps and a surgical blade or two forceps, discarding fly bodies when done.</li>
	<li>Use a small amount of PBS as a dissection buffer; desiccation must be avoided.</li>
	<li>Extract the full brain following the method of Williamson &#38; Hiesinger (2010)27, carefully removing cuticle particulates and any non-brain tissue (e.g. eye pigment tissue) (See Figure 1).</li>
	<li>Transfer the dissected brain into the appropriately-labeled well and repeat until there is a minimum of 20 brains per well.</li>
	<li>Keep plates on ice during dissection period and do not exceed longer than 20-30 min total time between the start of dissecting and assay initiation. 
	Note: More than 20 brains can be used so long as each group has the same number in each well. Minimum number of brains to achieve accurate reading may vary (refer to Discussion section for more detail).</li>
	<li>Once working set is complete, allow brains to incubate for 6 hr in 25 &#176;C with light shaking.</li>
	<li>Follow steps 1.2-1.6 for all remaining groups. 
	Note: Record and adjust end time of incubation depending on differentials between when each sample set was completed.</li>
</ol>
4. Preparation During Incubation Period
<ol>
	<li>Prepare the Modified Griess reagent as directed by the manufacturer (see table) and keep in the dark until use. The amount of reagent needed is dependent on the amount of samples. For example, the Griess reagent will be added in a 1:1 ratio to the Grace&#39;s Medium (step 5.5). Therefore, 50 &#956;l per sample set + 50 &#956;l for the blank control should be prepared.</li>
	<li>Construct a nitrite standard curve using sodium nitrite in a serial dilution ranging from 0.78125-100 mM nitrite for a total of 8 concentrations.&#160; 
	Note: This should be done for each experiment to ensure consistency and accuracy in sample concentration and Griess Reagent activity.</li>
	<li>Additionally, prepare and label sterile 0.5 ml centrifuge tubes, two tubes for each treatment group and two for the control.</li>
</ol>
5. Detection of Nitrite Levels
<ol>
	<li>Once the incubation period has ended, transfer the Grace&#39;s Medium (at least 45 &#956;l) from the microtiter wells into the first set of centrifuge tubes corresponding to each group. 
	Note: As previously noted, be sure to account for differences in incubation periods due to time taken between each set of dissections.</li>
	<li>Optional Step: Add nitrate reductase and NADPH at this step to convert nitrates to nitrites. (This step was not carried out on the results shown) 
	Note: A nitrate/nitrite assay may be conducted if desired with an aliquot of sample supernatant as described in assay kits.</li>
	<li>Gently centrifuge (around 8,175 x g) for 2 min to pellet any brain or tissue particulates that may have been aspirated from the wells.</li>
	<li>Pipette 30 &#956;l of the supernatant from the first set of centrifuge tubes into the second set of corresponding tubes, making sure not to disrupt the pellet after spinning down the samples.</li>
	<li>Add Griess reagent in a 1:1 ratio, incubate for 5-10 min and assay using NanoDrop spectrophotometer at 548 nm absorbance within 30 min. 
	Note: Samples can also be assayed using a microplate reader at an absorbance wavelength of 540 nm with 620 nm reference wavelength.</li>
	<li>Repeat each reading a minimum of three times and average the readouts. 
	Note: Be sure to &#34;blank&#34; the machine between every 6-10 readings and before completion, run the blank control (Grace&#39;s Medium plus Griess reagent without brains) as a sample to use as a standard deviation.</li>
	<li>Use the nitrite standard curve generated in step 4.2 to calculate relative nitrite levels of each sample.
	<ol>
		<li>To calculate unknown concentrations, first plot the values of the known concentrations from the standard curve using absorbance (i.e. optical density) on the y axis and concentration on the x axis.</li>
		<li>Once all standard curve values are graphed, find the slope of the best fit line and solve the equation for x. 
		Note: If serial dilutions are done properly, the best fit line should be linear or very close to it. If this is not the case, reconstruct standard curve. If problems persist, check all solutions, media and reagents for expiration and be sure equipment is working properly and within its range of detection.</li>
		<li>Next, simply substitute the absorbance values for y and solve for x, which represents concentration.</li>
	</ol>
	</li>
</ol>
For additional information regarding the use of NanoDrop equipment, refer to Desjardins et al.&#160;2009 and Desjardins &#38; Conklin, 201028,29.
6. Western Blot
<ol>
	<li>Feed 30 flies per condition as described above and decapitate heads, preferably using liquid nitrogen and vortexing.</li>
	<li>Homogenize whole heads on ice in 60 &#956;l RIPA buffer with 2 mM dithiothreitol (DTT) and 1x protease inhibitor cocktail added just before use.</li>
	<li>Spin down samples for 5 min at 8,175 x g in 4 &#176;C.</li>
	<li>Transfer supernatant to new centrifuge tubes.</li>
	<li>Mix samples gently and pipette 17 &#956;l of supernatant into centrifuge tubes containing 6.5 &#956;l 4x LDS sample buffer and 2.5 &#956;l 500 mM DTT. 
	Note: Flash frozen remaining sample in liquid nitrogen and stored in -20 &#176;C until use.</li>
	<li>Mix samples well and heat at 70 &#176;C for 10 min, mixing once midincubation.</li>
	<li>While heating, assemble polyacrylamide gel electrophoresis (PAGE) running apparatus using precast 4-12% NuPage Bis-Tris minigel, MOPS SDS running buffer with 500 &#956;l antioxidant in inner chamber.</li>
	<li>Quick spin samples and allow to cool.</li>
	<li>Load 25 &#956;l into each lane.</li>
	<li>Run the gel electrophoresis at 200 V on ice or in 4 &#176;C for 45 min or until gel is complete.</li>
	<li>Once electrophoresis is completed, transfer separated proteins from the gel to nitrocellulose membranes using iBlot Transfer Stacks and detect signal using Western Blot Detection Kit. 
	Note: Antibodies used are as followed: primary antibodies mouse &#945;-nNOS (1:250), mouse &#945;-syntaxin (1:200); secondary antibodies (1:250) provided in Western Blot Detection Kit (mouse).</li>
	<li>Detect chemiluminescent signal using western blot imaging system or X-ray film.</li>
</ol>
Note: Any standard immunoblot detection and quantification methods can be used for blot analysis.

<ol>
	<li>Gehrmann, J., Matsumoto, Y., Kreutzberg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+Intrinsic+immuneffector+cell+of+the+brain.">Microglia: Intrinsic immuneffector cell of the brain.</a> Brain Res. Rev. 20, 269-287 (1995).</li><li>Dissing-Olesen, 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=Axonal+lesion-induced+microglial+proliferation+and+microglial+cluster+formation+in+the+mouse.">Axonal lesion-induced microglial proliferation and microglial cluster formation in the mouse.</a> Neuroscience. 149, 112-122 (2007).</li><li>Aloisi, F., Penna, G., Cerase, J., Menendez Iglesias, B., Adorini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-12+production+by+central+nervous+system+microglia+is+inhibited+by+astrocytes.">IL-12 production by central nervous system microglia is inhibited by astrocytes.</a> J. Immunol. 159, 1604-1612 (1997).</li><li>Vincent, V. A., Tilders, F. J., Van Dam, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+endotoxin-induced+nitric+oxide+synthase+production+in+microglial+cells+by+the+presence+of+astroglial+cells:+a+role+for+transforming+growth+factor+beta.">Inhibition of endotoxin-induced nitric oxide synthase production in microglial cells by the presence of astroglial cells: a role for transforming growth factor beta.</a> Glia. 19, 190-198 (1997).</li><li>Rohrenbeck, A. 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=Upregulation+of+COX-2+and+CGRP+expression+in+resident+cells+of+the+Borna+disease+virus-infected+brain+is+dependent+upon+inflammation.">Upregulation of COX-2 and CGRP expression in resident cells of the Borna disease virus-infected brain is dependent upon inflammation.</a> Neurobiol. Dis. 6, 15-34 (1999).</li><li>Walsh, D. T., Perry, V. H., Minghetti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclooxygenase-2+is+highly+expressed+in+microglial-like+cells+in+a+murine+model+of+prion+disease.">Cyclooxygenase-2 is highly expressed in microglial-like cells in a murine model of prion disease.</a> Glia. 29, 392-396 (2000).</li><li>Aloisi, F., Serafini, B., Adorini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia-T+cell+dialogue.">Glia-T cell dialogue.</a> J. Neuroimmunol. 107, 111-117 (2000).</li><li>Minghetti, L., Levi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+as+effector+cells+in+brain+damage+and+repair:+focus+on+prostanoids+and+nitric+oxide.">Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide.</a> Prog. Neurobiol. 54, 99-125 (1998).</li><li>Mrak, R. E., Griffin, W. S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia+and+their+cytokines+in+progression+of+neurodegeneration.">Glia and their cytokines in progression of neurodegeneration.</a> Neurobiol. Aging. 26, 349-354 (2005).</li><li>Zhang, W., 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=Aggregated+alpha-synuclein+activates+microglia:+a+process+leading+to+disease+progression+in+Parkinson's+disease.">Aggregated alpha-synuclein activates microglia: a process leading to disease progression in Parkinson's disease.</a> FASEB J. 19, 533-542 (2005).</li><li>Beers, D. 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=Neuroinflammation+modulates+distinct+regional+and+temporal+clinical+responses+in+ALS+mice.">Neuroinflammation modulates distinct regional and temporal clinical responses in ALS mice.</a> Brain Behav. Immun. 25, 1025-1035 (2011).</li><li>Wood, W., Faria, C., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mechanisms+regulate+hemocyte+chemotaxis+during+development+and+wound+healing+in+Drosophila+melanogaster.">Distinct mechanisms regulate hemocyte chemotaxis during development and wound healing in Drosophila melanogaster.</a> J. Cell Biol. 173, 405-416 (2006).</li><li>Babcock, D. T., 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=Circulating+blood+cells+function+as+a+surveillance+system+for+damaged+tissue+in+Drosophila+larvae.">Circulating blood cells function as a surveillance system for damaged tissue in Drosophila larvae.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 10017-10022 (2008).</li><li>Vlisidou, I., 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+embryos+as+model+systems+for+monitoring+bacterial+infection+in+real+time.">Drosophila embryos as model systems for monitoring bacterial infection in real time.</a> PLoS Pathog. 5, e1000518 (2009).</li><li>Foley, E., O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+contributes+to+induction+of+innate+immune+responses+to+gram-negative+bacteria+in+Drosophila.">Nitric oxide contributes to induction of innate immune responses to gram-negative bacteria in Drosophila.</a> Genes Dev. 17, 115-125 (2003).</li><li>Costa, A., Jan, E., Sarnow, P., Schneider, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Imd+pathway+is+involved+in+antiviral+immune+responses+in+Drosophila.">The Imd pathway is involved in antiviral immune responses in Drosophila.</a> PloS One. 4, e7436 (2009).</li><li>Daigle, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+microglial-like+neuroinflammatory+and+hypoxia+responses+in+a+Drosophila+model+for+Parkinson's+disease.">Integrated microglial-like neuroinflammatory and hypoxia responses in a Drosophila model for Parkinson's disease.</a> , .</li><li>Tanner, C. M., Kamel, F., Ross, G. W., Hoppin, J. A., Goldman, S. M., Korell, M., Marras, C., Bhudhikanok, G. S., Kasten, 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=Rotenone,+Paraquat+and+Parkinson's+Disease.">Rotenone, Paraquat and Parkinson's Disease.</a> Environ. Health Perspect. 119 (6), 866-8672 (2011).</li><li>Manning-Bog, A. B., McCormack, A. L., Li, J., Uversky, V. N., Fink, A. L., Di Monte, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+herbicide+paraquat+causes+up-regulation+and+aggregation+of+alpha-+synuclein+in+mice:+paraquat+and+alpha-synuclein.">The herbicide paraquat causes up-regulation and aggregation of alpha- synuclein in mice: paraquat and alpha-synuclein.</a> J. Biol. Chem. 277, 1641-1644 (2002).</li><li>Ossowska, K., Wardas, J., Smialowska, M., Kuter, K., Lenda, T., Wieronska, J. M., Zieba, B., Nowak, P., Dabrowska, J., Bortel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+slowly+developing+dysfunction+of+dopaminergic+nigrostriatal+neurons+induced+by+long-term+paraquat+administration+in+rats:+An+animal+model+of+preclinical+stages+of+Parkinson's+disease.">A slowly developing dysfunction of dopaminergic nigrostriatal neurons induced by long-term paraquat administration in rats: An animal model of preclinical stages of Parkinson's disease.</a> Eur. J. Neurosci. 22, 1294-1304 (2005).</li><li>McCormack, 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=Role+of+oxidative+stress+in+paraquat-induced+dopaminergic+cell+degeneration.">Role of oxidative stress in paraquat-induced dopaminergic cell degeneration.</a> J. Neurochem. 93, 1030-1037 (2005).</li><li>Chaudhuri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+genetic+and+environmental+factors+in+a+Drosophila+parkinsonism+model.">Interaction of genetic and environmental factors in a Drosophila parkinsonism model.</a> J. Neurosci. 27, 2457-2467 (2007).</li><li>Krishnakumar, S., Burton, D., Rasco, J., Chen, X., O'Donnell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+GTP+cyclohy-+drolase+I+and+tyrosine+hydroxylase+in+Drosophila.">Functional interactions between GTP cyclohy- drolase I and tyrosine hydroxylase in Drosophila.</a> J. Neurogenet. 14 (1), 1-23 (2000).</li><li>Foxton, R. H., Land, J. M., Heales, S. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrahydro-+biopterin+availability+in+Parkinson's+and+Alzheimer's+disease;+potential+pathogenic+mechanisms.">Tetrahydro- biopterin availability in Parkinson's and Alzheimer's disease; potential pathogenic mechanisms.</a> Neurochem. Res. 32 (4-5), 751-756 (2007).</li><li>Inamdar, A. A., Chaudhuri, A., O'Donnell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Protective+Effect+of+Minocycline+in+a+Paraquat-Induced+Parkinson's+Disease+Model+in+Drosophila+is+Modified+in+Altered+Genetic+Backgrounds.">The Protective Effect of Minocycline in a Paraquat-Induced Parkinson's Disease Model in Drosophila is Modified in Altered Genetic Backgrounds.</a> Parkinson's Disease. 2012, 938528 (2012).</li><li>Kean, L., Cazenave, W., Costes, L., Broderick, K. E., Graham, S., Pollock, V. P., Davies, S. A., Veenstra, J. A., Dow, 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=Two+nitridergic+peptides+are+encoded+by+the+gene+capability+in+Drosophila+melanogaster.">Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster.</a> Am. J. Phys. Reg. Integr. Comp. Phys. 282, R1297-R1307 (2002).</li><li>Williamson, W. R., Hiesinger, P. R. <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+Developing+and+Adult+Drosophila+Brains+and+Retinae+for+Live+Imaging.">Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging.</a> J. Vis. Exp. (37), (2010).</li><li>Desjardins, P., Hansen, J. B., Allen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvolume+Protein+Concentration+Determination+using+the+NanoDrop+2000c+Spectrophotometer.">Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer.</a> J. Vis. Exp. (33), (2009).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+Microvolume+Quantitation+of+Nucleic+Acids.">NanoDrop Microvolume Quantitation of Nucleic Acids.</a> J. Vis. Exp. (45), (2010).</li></ol>

The authors declare no competing financial interests.

This method, though simple in approach, provides a cost-efficient, highly repeatable and exceedingly sensitive method for quantifying levels of NOS-mediated neuroinflammation. As shown in the Results section, there are many variables that can be adjusted to enhance or optimize the response in different induction models or organisms. Because this is a highly sensitive system, however, these variables may also result in highly variable outcomes if all aspects of the protocol are not handled carefully. When first testing using any model, we recommend setting up the parameters by testing variables, which include chemical concentration (Figure 4A), length of exposure (Figures 5A and&#160;5B), and incubation duration (Figure 5C). Be mindful of other variables, such as the number of brains/tissue samples needed to achieve detection threshold and consistency, even when applying this method to similar Drosophila models. Additionally, in age-dependent genetic models of inflammation, extended time point optimization will be needed.
As mentioned above, due to the sensitive nature of the assay, inconsistencies in the protocol can yield unreliable results. With this in mind, all equipment and materials were sterilized prior to use. Due to the short incubation time, no antibiotics were added to the culture media in these experiments. However, if the protocol is adapted for longer incubation times or if bacterial growth is observed in cultures or media, typical culture antibiotics such as penicillin-streptomycin should be added and equipment should be re-sterilized. As in any chemical treatment experiments caution should be used to ensure that all groups are reared under well-controlled conditions, gender and age matched, and all groups receive exactly the same feeding conditions. Some example feeding conditions to consider include variations in concentrations, availability and access to feeding source, duration of feeding and the time of the day that the feeding is administered. Detailed records for all experimental conditions and close attention to the natural feeding patterns of your model will help minimize variability. If mutant or transgenic strains that affect neural circuitry or behavioral patterns are employed an important control would include monitoring of feeding behavior.
Once again, while similar protocols are available in cell culture and lysates, one major advantage of this method is that the integrity of the tissue is maintained, therefore allowing virtually all cellular relationships within the brain to remain intact. This system represents a relatively natural, in vivo state of neuroinflammatory signaling and cellular secretion. Established in Drosophila, this technique can be easily tailored to other small model organisms, and is not limited to the central nervous system, as demonstrated by a similar application for NO signaling in a Malpighian tubule model for kidney function26. The most significant advantage by far of this protocol is the ability to manipulate and adapt this assay to fit the needs and interests of the researcher or model system.
Through the use of combination chemical-genetic approaches, this assay has the potential to significantly enhance the ability of small model systems to investigate the mechanisms, genetic components and chemical modulators of inflammation.

Neuroinflammation is an intricate immune response that has been shown to be intimately associated with the pathology of a wide range of diseases, the majority of which are neurodegenerative disorders. Under basal conditions, this multifaceted network of peripatetic immune cells maintains tissue health through its rapid response to infection, plaques, or injury1,2. A neuroinflammatory response by the mammalian central nervous system can be triggered due to minor insults, such as cellular oxidative stress associated with normal aging, or major assaults, such as neuron damage occurring from acute exposure to a chemical toxin. When this induction occurs, phagocytic surveillance cells known as microglia are activated and migrate to the site of neuronal cellular damage. These scavenger-like immune cells, which are similar to macrophage cells that exists in the periphery, then begin a sophisticated signaling cascade with the damaged neuron, as well as with astrocytes, supporting glial cells that act alongside microglia for the typically robust microglial response3-7. Once activated, the microglia begin a process of inflammation to stimulate the immune system and promote tissue repair1,8.
Under conditions of sustained insult, as in neurodegenerative diseases such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and amyotrophic lateral sclerosis, chronic inflammation causes significant damage to healthy tissue as well. Recent studies have shown that this persistent, aggressive response by the immune system may in fact be leading to an over-excitatory reaction that exacerbates the disease state rather than ameliorating the condition9-11.
In Drosophila melanogaster, the innate immune response is genetically well-conserved. Although lacking a true adaptive immune network, Drosophila maintains a sophisticated innate immune system containing a class of phagocytes known as hemocytes. These immune cells have been previously identified as macrophage-like cells that migrate to the site of injury12,13, and undergo phagocytosis during septic shock14, introduction of a parasite15, or viral infection16.
Importantly, like mammalian macrophages, phagocytic hemocytes express nitric oxide synthase (NOS) and produce nitric oxide (NO), a critical signaling component of Drosophila immunity15,16. All of these studies, however, have only been able to demonstrate hemocyte responses during embryonic development and in larva.
Our lab has observed a robust, hemocyte-mediated neuroinflammation in Drosophila adult brains, a previously unknown phenomenon17. This response was induced through exposure to the toxicant paraquat. Epidemiological studies by the National Institutes of Health have identified the widely-used herbicide as a potential risk factor in the onset of PD in humans18. Research investigating paraquat treatment in mammalian models had already validated its neurotoxic properties and had shown the herbicide to result in Parkinsonian symptoms, such as motor abnormalities and selective neuron loss19-21. In Drosophila, treatment of paraquat leads to a wide range of Parkinsonian behavioral phenotypes as well as dopaminergic neuron death consistent with PD pathology22. Though the mechanisms and genetic factors associated with this neuroinflammatory response have not yet been fully elucidated in Drosophila, we have observed a conserved induction of NOS during neurodegeneration.
Using this method, we have additionally detected a similar, albeit less dramatic, neuroinflammatory response using mutant genotypes with known neurotoxic sensitivity. Encoded by the gene Punch, GTP cyclohydrolase is the first and rate-limiting protein in the biosynthesis of BH4, which then acts as a necessary cofactor for NOS23,24. The loss-of-function mutation PuZ22 causes a decline in the inflammatory response and heightened susceptibility to paraquat25. With this protocol, we were able to quantify relatively subtle modifications of nitric oxide levels induced by this genetic variation.
To detect NO in our samples we use Griess reagent, a widely-accepted colorimetric method for measuring NO levels. This reagent indirectly measures relative NO abundance by detecting the presence of nitrites, one of two end products of nitric oxide production. The existing nitrite detection protocols using Griess reagent have been applied primarily for in vitro analyses or in cell culture, where NO diffuses into the culture medium. We initially developed a Griess reagent-based method for detecting NOS in crude Drosophila head homogenates25. We have, however, found that method to be somewhat variable with altered conditions, presumably due to the instability, high reactivity and low relative concentration of NO in whole head tissues. Therefore, we sought to develop a method based on cell culture protocols that would allow for greater sensitivity and reliability in quantifying NOS activity. Here we describe a method conceptually similar to one previously employed to detect NOS in Drosophila larval Malpighian tubules26. In this procedure, we utilize whole brains that were dissected immediately following a toxin treatment to induce the inflammatory response. Then, we incubated these samples in insect culture medium to preserve the integrity of the tissue, and to enhance the sensitivity and reliability of a Griess reagent-based assay.
We anticipate that, with the use of the sophisticated genetic tools available, this model has the capacity to serve as a promising utility for investigating the dynamic inflammation/neurodegeneration network. With the ability to accurately quantify the activation and relative intensity of the neuroinflammatory response, this novel assay creates a primary tissue culture capable of detecting secreted neuroinflammatory markers. This method offers small organism models of inflammation an inexpensive, highly sensitive technique for rapidly assaying whole-organism or live-tissue samples where previously lacking.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Feeding Chambers</td>
		</tr>
		<tr>
			<td>Empty Drosophila vial</td>
			<td>Genesee Scientific</td>
			<td>#32-116</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent cotton</td>
			<td></td>
			<td></td>
			<td>Any standard product can be used.</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher</td>
			<td>#09-802-1B</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Paraquat exposure</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>#S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraquat (methyl viologen dichloride hydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>#856177</td>
			<td>Toxic. Handle with Care and within MSDS guidelines</td>
		</tr>
		<tr>
			<td>L-NAME (N-Nitro-L-arginine methyl ester hydrochloride)</td>
			<td>Sigma-Aldrich</td>
			<td>#N5751</td>
			<td></td>
		</tr>
		<tr>
			<td>D-NAME (N-Nitro-D-arginine methyl ester hydrochloride)</td>
			<td>Sigma-Aldrich</td>
			<td>#N4770</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Dissecting Adult Fly Brains and Incubation</td>
		</tr>
		<tr>
			<td>Grace&#39;s Insect Medium</td>
			<td>Invitrogen</td>
			<td>#11605-094</td>
			<td>Grace&#39;s Medium may also be substituted for Sf-900 II SFM or Sf-900 III SFM Medium for higher lot-to-lot consistency.</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>#3596</td>
			<td>Any standard product can be used.</td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td></td>
			<td></td>
			<td>Any standard product can be used.</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>#0197</td>
			<td>Any standard product can be used.</td>
		</tr>
		<tr>
			<td>Medical Tweezers 5</td>
			<td>Dumont</td>
			<td>EMS #72877-D</td>
			<td>Any standard product can be used.</td>
		</tr>
		<tr>
			<td>10x PBS buffer</td>
			<td>OmniPur</td>
			<td>#6508</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Detection of Nitrite Levels</td>
		</tr>
		<tr>
			<td>Modified Griess reagent</td>
			<td>Sigma-Aldrich</td>
			<td>#4410</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Sigma-Aldrich</td>
			<td>#S2252 or #237213</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Assay Kit</td>
			<td>Bio-Rad</td>
			<td>500-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td></td>
			<td></td>
			<td>Any standard product can be used.</td>
		</tr>
		<tr>
			<td>Nitrate reductase</td>
			<td></td>
			<td></td>
			<td>optional</td>
		</tr>
		<tr>
			<td>NADPH</td>
			<td></td>
			<td></td>
			<td>optional</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Western Blot Analysis</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td>Amresco</td>
			<td>97063-270</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol (DTT)- Reducing agent</td>
			<td>Novex</td>
			<td>B0009</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Amresco</td>
			<td>97063-082</td>
			<td></td>
		</tr>
		<tr>
			<td>4x LDS sample buffer</td>
			<td>Novex</td>
			<td>B0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Precast 4-12% Bis-Tris minigel</td>
			<td>Novex</td>
			<td>BG04125BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS SDS running buffer</td>
			<td>Novex</td>
			<td>B0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Antioxidant</td>
			<td>NuPage</td>
			<td>NP0005</td>
			<td></td>
		</tr>
		<tr>
			<td>iBlot Transfer Stacks (Nitrocellulose)</td>
			<td>Invitrogen</td>
			<td>IB301002</td>
			<td></td>
		</tr>
		<tr>
			<td>Western blot detection kit</td>
			<td>Invitrogen</td>
			<td>IB711002</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse &#945;-nNOS</td>
			<td>BD Biosciences</td>
			<td>611853</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse &#945;-syntaxin</td>
			<td>DSHB</td>
			<td>8C3</td>
			<td></td>
		</tr>
	</tbody>
</table>

novel whole-tissue quantitative assay of nitric oxide levels in drosophila neuroinflammatory response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Paraquat has been shown in numerous animal models, such as mice19, rats20 and fruit flies22, to induce neurological degeneration consistent with Parkinsonian-like pathology. We have previously reported that the anti-inflammatory antibiotic minocycline, when cofed with paraquat, results in extended survival, reduced production of reactive oxygen species and rescue of dopaminergic neuron death25. Therefore, as minocycline acts to suppress inflammation, we began investigating NOS, a key protein in the activation of the inflammatory response, and its role in modulating paraquat toxicity.
A paraquat toxicity curve was performed to establish an effective lethal dose and toxic treatment range, using paraquat concentrations between 1.25 and 10 mM (Figure 2A). We also assayed the effects of paraquat concentrations of 20 mM and 40 mM, but found that toxicity was too acute at these higher concentrations to accurately assess cellular responses to this oxidative stressor, and therefore, we have not included the results for these concentrations in this report. Using L-NAME, a competitive NOS inhibitor, and D-NAME, the inactive isomer of NAME, we cotreated adult male flies with paraquat and one of the NAME isomers. Cotreatment of flies with paraquat and L-NAME resulted in a significant rescue of lifespan truncation caused by paraquat ingestion, while flies cotreated with paraquat and D-NAME showed no improvement in survival (Figure 2B), supporting the hypothesis that suppressing inflammation through the inhibition of NOS enhanced survival of paraquat-treated flies.
As further validation of a NOS-mediated paraquat response, we detected changes in NOS protein levels directly after paraquat exposure. NOS protein levels increased in a paraquat concentration-dependent manner (Figure 3A). When treating flies with 10 mM paraquat over exposure durations ranging from 6-30 hr, we observed an initial increase, then a decrease as exposure time increased (Figure 3B), consistent with the patterns of nitrite levels observed in our variant of the Griess assay (Figure 5B). These results are consistent with our reports that paraquat treatment causes induction of NOS and that inhibition of NOS or treatment with L-NAME or minocycline provides partial protection17,25.
Figure 4A demonstrates a linear relationship between the increase in paraquat concentration and the magnitude of the inflammatory response as defined by the secretion and detection of NO. Under basal levels, heterozygous Punch mutants secreted slightly lower levels of NO, a variation too indiscernible to be determined using previous detection methods. When fed paraquat, a pronounced increase in NO levels were observed in the Punch mutant, however, significantly less than the wildtype treated flies (Figure 4B). The relative values of the wildtype and Punch flies can be accurately and reproducibly quantified, though the variation at untreated levels were extraordinarily subtle.
When working with toxins or chemicals, it is vital to establish both a time-of-exposure curve as well as a concentration-based toxicity curve, since these conditions can dramatically alter the sensitivity of the assay. For example, treatment with 5 mM paraquat results in maximum NO detection after 24 hr of exposure (Figure 5A), while exposure to 10 mM paraquat resulted in more rapid induction (maximum levels at 12 hr), but also more rapid decay of activity during extended exposures (Figure 5B). Although a higher concentration with quick induction might seem preferable, a maximized response accelerates death of both neurons and possibly hemocytes to the point that reproducibility may become difficult.
NO molecules are unstable and highly reactive. A successful assay must therefore include incubation conditions that provide an optimal balance between continued induction of NOS and rapid turnover of NO. In order to define optimal incubation conditions, we assessed the effect of the time that dissected brains were incubated in the culture medium prior to adding the Griess reagent. We found that a 6 hr incubation at room temperature produced maximum nitrite levels in the subsequent Griess reaction (Figure 5C) .We expect that variations in the incubation period may be needed to optimize detection for various models of inflammation. Careful establishment of all optimal parameters for the particular organism, genotype, and tissue being assayed, will be essential to achieve reliable and accurate results since the process is highly dynamic. In particular, genetic variants and transgene expression models are expected to require substantial optimization, particularly with respect to developmental stage or age of adults.
<img alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50892/50892fig1highres.jpg" src="/files/ftp_upload/50892/50892fig1.jpg" /> 
Figure 1. Drosophila adult brain. Light microscopy of dissected adult male brain 2-3 days posteclosion. MB = midbrain (central cortex), OL = optic lobe.
<img alt="Figure 2" fo:content-width="4.5in" fo:src="/files/ftp_upload/50892/50892fig2highres.jpg" src="/files/ftp_upload/50892/50892fig2.jpg" /> 
Figure 2. Paraquat results in a dose-dependent reduction in lifespan, mediated through nitric oxide synthase. Wild type male flies fed (A) 5% sucrose (control), and serial dilution of paraquat ranging from 1.25-10 mM concentration in 5% sucrose and (B) cofed paraquat with L-NAME or D-NAME for 10 consecutive days. N = 3 groups of 10. Error bars = SEM.
<img alt="Figure 3" src="/files/ftp_upload/50892/50892fig3.jpg" /> 
Figure 3. Nitric oxide synthase protein levels increase with paraquat concentration and length of exposure. Immunoblot detection of nitric oxide synthase levels of flies treated with varying (A) paraquat concentrations (12 hr exposure) and (B) 10 mM paraquat with varying exposure durations. Protein levels were detected on nitrocellulose membranes using chemiluminescence and X-ray film exposure.
<img alt="Figure 4" fo:content-width="4.5in" fo:src="/files/ftp_upload/50892/50892fig4highres.jpg" src="/files/ftp_upload/50892/50892fig4.jpg" /> 
Figure 4. Nitric oxide detection levels after paraquat exposure. Male wild type (WT) flies 2-3 days posteclosion were assayed following 12 hr feeding. (A) Concentration curve of paraquat concentrations. (B) Male Punch heterozygous mutants fed sucrose control and 10mM paraquat. N = 20 brains/group. Results represent 3 replications per group. Error bars = SEM. Statistical analyses were performed using one-way ANOVA with Dunnett&#39;s Multiple Comparison posttest (*, p &#60; 0.05, **, p &#60; 0.01, ***, p &#60; 0.001).
<img alt="Figure 5" fo:content-width="4.5in" fo:src="/files/ftp_upload/50892/50892fig5highres.jpg" src="/files/ftp_upload/50892/50892fig5.jpg" /> 
Figure 5. Optimizing the detection of nitric oxide production by altering the length of paraquat exposure and time of tissue incubation. Relative nitric oxide levels on male brains 2-3 days posteclosion following 12 hr treatment of (A) 5 mM paraquat and (B) 10 mM paraquat. (C) NO curve using varying tissue incubations periods following 12 hr, 10 mM paraquat feeding. Significance values were calculated relative to the following: (A, B) 5% sucrose controls for each exposure time (not shown in graph), (C) 5% sucrose controls for each incubation time (not shown in graph). N = 20 brains/group. Results represent 3 replications per group. Error bars = SEM. Statistical analyses were performed using one-way ANOVA with Dunnett&#39;s Multiple Comparison posttest (*, p&#60;0.05, **, p&#60;0.01, ***, p&#60;0.001).

Watch this Scientific Journal Video about Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response at JoVE.com

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

novel-whole-tissue-quantitative-assay-nitric-oxide-levels-drosophila

Research

JoVE Journal

Immunology and Infection

7.9K Views.  University of Alabama. Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Video: Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.