Name: high and low throughput screens with root-knot nematodes meloidogyne spp.
Uploaded: 2012-03-12T12:00:00
Duration: 11 min 46 s
Description: Watch this Scientific Journal Video about High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp. at JoVE.com

Research in Kaloshian lab is funded by a grant from United States National Institute of Food and Agriculture (2007-35607-17765). Research in Roberts lab is funded by grants from United States Agency for International Development (GDG-G-00-02-00012-00 and EDH-A-00-07-00005) and the California Dry Bean Advisory Board.

1. Tomato Seedling Growth in Pots and Trays
<ol>
 <li>For pot assays, plant tomato seeds in a single pot in an organic-rich soil such as Sunshine mix. Maintain in a greenhouse at 22-28&deg;C. After germination, fertilize once a week with Miracle-Gro.</li>
 <li>About two weeks after germination, at the two true-leaf stage, transplant individually the seedlings into pots (10 cm diameter and 17 cm deep) filled with sterile sandy soil containing 90% sand and 10% organic mix. Add Osmocote slow release fertilizer and maintain in a greenhouse at 22-28&deg;C for two weeks. Continue to fertilize plants once a week with Miracle-Gro.</li>
 <li>For high-throughput screens, plant seeds directly in trays in sandy soil, cover the tray with plastic wrap until germination and maintain as above. After germination, add Osmocote slow release fertilizer and fertilize once a week with Miracle-Gro.</li>
</ol>
2. Cowpea Growth in Seedling Pouches
<ol>
 <li>Seeds are either germinated in a Petri dish, lined with several layers of Kimwipe paper, and transferred singly to pouches or placed directly into the paper groove of the pouch and germinated.</li>
 <li>Place the pouches in a plastic hanging file folder, two per folder, and arrange the folders in a rack in a vertical position. Place the rack in a controlled-environment chamber maintained at a temperature of 25-28 &deg;C and 16 h light / 8 h dark cycle. Racks also can be maintained in a greenhouse, but require a foil or stiff paper cover placed over the rack on both sides of the plant stems, to reduce potential for fungal contamination.</li>
 <li>Water the pouches once or twice per day with reverse osmosis water. About 10 to 14 days after seeding, when an adequate root system with tertiary root tips has developed (Figure 1), the seedlings are ready for inoculation.</li>
</ol>
3. Extraction of Nematode Eggs
Extraction of eggs from infected roots is modified from a protocol developed by Hussey and Barker (1973).
<ol>
 <li>Three to four days before nematode inoculation, extract root-knot nematode eggs from infected tomato roots. Before starting egg extraction, wash the work area thoroughly with hot water to avoid contamination. Also wash in hot water a blender, two buckets, a wire mesh support, a rubber mallet, three sieves of 425, 90 and 25 &mu;m aperture, a graduated cylinder and scissors.</li>
 <li>Stack the sieves from top to bottom in the following order: 425, 90 and 25 &mu;m aperture. The eggs will be collected on the 25 &mu;m sieve at the bottom. Put the sieves on a wire mesh supported in a sink. Put a bucket under the wire mesh to collect the run-through solution.</li>
 <li>Cut the tops of the infected plant(s), used as the source of the inoculum, and discard. Carefully remove the plant from the pot. Wash the roots by dipping in a plastic bucket full of water. Rinse the roots further under running water until the soil particles are washed away from the roots.</li>
 <li>Cut the roots with scissors into small pieces. Dispose of the taproot.</li>
 <li>Put the chopped roots from a single plant in a large plastic jar with a lid, add just enough 10% bleach solution to cover the roots and close the lid.</li>
 <li>Shake the jar containing the roots for 2 min.</li>
 <li>Open the jar and pour the roots onto the top sieve and wash with a hose attached to a misting nozzle. Wash well until all the bleach smell is gone. Use a mallet to tap the sides of the sieves to avoid clogging the sieve pores.</li>
 <li>Remove the top sieve and rinse the debris on the second sieve.</li>
 <li>Remove the second sieve and collect the fine debris, which includes the eggs, from the last sieve. With a gentle stream from a water bottle, move the debris to one side of the sieve. Collect the debris and eggs into a clean beaker with minimum amount of water.</li>
 <li>Sieve once more the water collected in the bucket through the 25 &mu;m sieve to collect any eggs that may have escaped. Rinse well with water and collect in the same beaker.</li>
 <li>Discard the plant debris from the top sieve and wash thoroughly all three sieves with pressurized water. Do not touch the mesh part of the sieves as it may distort the pore size.</li>
</ol>
4. Hatching Nematode Eggs
<ol>
 <li>Line a clean metal basket with a few layers of Kimwipe paper and fit on a glass Petri dish. Allow a 1-cm distance between the bottom of the basket and the dish.</li>
 <li>Pour the extracted nematode eggs onto the paper in the wire basket. Add enough liquid so that the bottom of the wire basket touches the water surface but is not immersed in water. Cover the top with a plastic lid.</li>
 <li>Every day check for water evaporation and add some water in the Petri dish so that the bottom of the basket touches the water. This will prevent the eggs from drying out.</li>
 <li>Every other day, collect the water that contains the J2s from the Petri dishes into a beaker. If not used immediately, aerate the collected inoculum at room temperature using a lab air supply or air generated by an aquarium pump. Use the aerated inoculum within 2 days. J2s may be collected from the hatching set up over a period of 6-8 days.</li>
</ol>
5. Tomato Root Infection in Pots or Trays and Evaluation of Infection
<ol>
 <li>Use three aliquots of the collected nematodes to count the number of J2s in a counting slide or dish, calculate the average and determine the desired volume for inoculation. Typically 3000 J2s are used per plant in pots and 500 J2s for plants grown in seedling trays.</li>
 <li>Stir the inoculum on a magnetic stirring plate at low speed.</li>
 <li>Before you inoculate the plants, make sure that the soil is moist but not too wet. For pot inoculation, make three holes of about half-pot depth in the sand around each tomato root system using a pencil (Figure 2). Inoculate each plant by delivering the J2s into the three holes using a pipette. Afterwards, cover the holes.</li>
 <li>For the high-throughput screens in trays, use a modified sealed tip needle with holes on the sides glued to a 5-ml pipette tip along a pipetter (Figure 3) to deliver the J2s into the soil. Alternatively, nematodes can be delivered as in step 5.3.</li>
 <li>Maintain the plants in a greenhouse at 24-27&deg;C for six to eight weeks. Continue to fertilize twice a month with Miracle-Gro.</li>
 <li>For evaluation of infection, carefully remove the plants from the pots and wash the roots (as described in step 3.3).</li>
 <li>Stain the egg masses blue by submerging the roots in 1 mg/L erioglaucine, for 15 min.</li>
 <li>Rinse the roots in water and evaluate by counting the stained egg masses on individual root system. An illuminated desk magnifier can be used to help visualize the egg masses.</li>
 <li>If the screened plants are needed for further genetic studies, do not cut the tops before washing the roots for evaluation. After staining and counting the egg masses, transplant the seedlings in organic soil, prune the tops heavily to reduce transpiration, and maintain in a greenhouse.</li>
</ol>
6. Cowpea Root Infection in Pouches and Evaluation of Infection
<ol>
 <li>Count the nematode inoculum and stir on a magnetic stirring plate as described earlier.</li>
 <li>Remove the pouches from the hanging folders and place on a horizontal surface. Inoculate each pouch with 1500 J2s in 5 ml. Lift the plastic cover of the pouch and distribute the nematodes evenly over the surface of the roots.</li>
 <li>Keep the pouches in a horizontal position for 24 h after inoculation, covered with dark paper to exclude light, then return to the hanging folders in the growth chamber.</li>
 <li>Water the plants as needed, once or twice daily, with half-strength Hoagland's solution (Hoagland &amp; Arnon, 1950; Table 1) until a response to the fertilizer is observed, typically enhanced foliage greening and more vigorous shoot growth. Usually the plants are watered with half-strength Hoagland 3 days in a row. Afterwards, keep the pouches moist with water.</li>
 <li>Approximately 30 days (range 28-35 days) after inoculation, infuse each pouch with about 10-20 ml of 75 mg/L erioglaucine. Keep the pouches flooded with the dye in a horizontal position overnight.</li>
 <li>Drain the pouches and evaluate the root systems by counting the egg masses under an illuminated desk magnifier.</li>
 <li>If the screened plants are needed for further genetic studies or breeding work, carefully pull out the roots from the paper, transplant in organic soil in pots and maintain in a greenhouse.</li>
</ol>
7. Representative Results
The appropriate stages of tomato and cowpea plants for nematode inoculations for the two systems described are shown in Figures 1 and 2. In addition, examples of well-infected roots of tomato and cowpea are shown in Figures 4 and 5. As in most disease resistance screens, it is advisable to use at least 6-10 plants per genotype for nematode inoculation to calculate the average of the infection rate. Variation in nematode infection between plants, of the same genotype, can be reduced by using uniform plant size and accurate amount and delivery of inoculum.
The use of trays and growth pouches enables screening of hundreds to thousands of plants in a small growth space. Growth pouches also allow fast and efficient non-destructive evaluation of root-knot nematode infections with no need for washing roots (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/3629/3629fig1.jpg" /> 
Figure 1. A two week-old cowpea plant grown in a pouch and ready for root-knot nematode inoculation.
<img alt="Figure 2" src="/files/ftp_upload/3629/3629fig2.jpg" /> 
Figure 2. A three week-old tomato plant ready for root-knot nematode inoculation.
<img alt="Figure 3" src="/files/ftp_upload/3629/3629fig3.jpg" /> 
Figure 3. A modified needle and pipette tip used for high-throughput inoculation of nematodes. The bottom of a needle was sealed and three sets of holes were drilled in the needle using a laser beam. Then, the modified needle was glued to a 5-ml pipette tip.
<img alt="Figure 4" src="/files/ftp_upload/3629/3629fig4.jpg" /> 
Figure 4. A tomato root system infected with root-knot nematodes.
<img alt="Figure 5" src="/files/ftp_upload/3629/3629fig5.jpg" /> 
Figure 5. A cowpea root system with egg masses stained with erioglaucine 30 days post-inoculation grown at 28 &deg;C.

<ol>
	<li>Bhattarai, K. K., Xie, Q. G., Mantelin, S., Bishnoi, U., Girke, T., Navarre, D. A., Kaloshian, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+susceptibility+to+root-knot+nematodes+requires+an+intact+jasmonic+acid+signaling+pathway.">Tomato susceptibility to root-knot nematodes requires an intact jasmonic acid signaling pathway.</a> Mol. Plant Microbe Interact. 21, 1205-1214 (2008).</li><li>Ehlers, J. D., Matthews, W. C., Hall, A. E., Roberts, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+a+broad-based+form+of+root-knot+nematode+resistance+in+cowpea.">Inheritance of a broad-based form of root-knot nematode resistance in cowpea.</a> Crop Sci. 40, 611-618 (2000).</li><li>Hoagland, D. R., Arnon, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+water-culture+method+for+growing+plants+without+soil.">The water-culture method for growing plants without soil.</a> Calif. Agric. Exp. Stn. Circ. 347, (1950).</li><li>Hussey, R. S., Barker, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+methods+of+collecting+inocula+of+Meloidogyne+spp.,+including+a+new+technique.">A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique.</a> Plant Dis. Rep. 57, 1025-1028 (1973).</li><li>Martinez de Ilarduya, O., Moore, A. E., Kaloshian, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tomato+Rme1+locus+is+required+for+Mi-1-mediated+resistance+to+root-knot+nematodes+and+the+potato+aphid.">The tomato Rme1 locus is required for Mi-1-mediated resistance to root-knot nematodes and the potato aphid.</a> Plant J. 27, 417-425 (2001).</li><li>Matkin, O. A., Chandler, P. A., Baker, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+U.C.-type+soil+mixes.+In:+The+U.C.+system+for+producing+healthy+container-grown+plants.">The U.C.-type soil mixes. In: The U.C. system for producing healthy container-grown plants.</a> Calif. Agric. Exp. Stn. Manual. 23, 68-85 (1957).</li><li>Omwega, C. O., Roberts, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+resistance+to+Meloidogyne+spp.+in+common+bean+and+the+genetic+basis+of+its+sensitivity+to+temperature.">Inheritance of resistance to Meloidogyne spp. in common bean and the genetic basis of its sensitivity to temperature.</a> Theor. Appl. Genet. 83, 720-726 (1992).</li><li>Omwega, C. O., Thomason, I. J., Roberts, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nondestructive+technique+for+screening+bean+germplasm+for+resistance+to+Meloidogyne+incognita.">A nondestructive technique for screening bean germplasm for resistance to Meloidogyne incognita.</a> Plant Dis. 72, 970-972 (1988).</li><li>Perry, R. N., Moens, M., Starr, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Root-Knot Nematodes. , (2009).</li><li>Starr, J. L., Cook, R., Bridge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Resistance to Parasitic Nematodes. , (2002).</li></ol>

We have nothing to disclose.

There are two critical steps for a successful nematode screen: preparation of a highly infective inoculum and using plants at the proper developmental stage. Hatching rate of root-knot nematode eggs is highly variable and ranges between 5- 50%. Therefore to obtain optimum levels of hatch and highly infective J2s, close attention must be paid to the egg extraction and hatching procedures. Eggs should be exposed to bleach for a minimum time period and bleach should be rinsed out well from the root mixture. When hatching the eggs, the slurry containing the eggs should not be submerged in water. In addition, avoid spilling the eggs into the underlying Petri dish where the hatched juveniles are collected. One way to avoid spilling the eggs during the hatching process is not to add water directly to the Kimwipe as the paper might break. Instead add the water directly to the Petri dish.
For best results, use freshly hatched J2s. If the hatch rate is low and more inoculum is needed, J2s can be stored at 15&deg;C for a longer period. Warm the stored inoculum to room temperature to revive the nematodes prior to inoculation. However, do not store the J2s for prolonged periods even at 15&deg;C as starved J2s will not infect efficiently.
Young seedlings are the best developmental stage for root-knot nematode inoculation. However, this needs to be balanced with the formation of an adequate root system providing sufficient numbers of root tips as points of entry for the J2s. Maintaining optimal plant growth condition is also critical for the screens. Avoid over-watering plants specially those grown in pouches. Over-watering the pouches, as indicated by standing water in the bottom of the pouch, can promote fungal growth and diminish root health.
With both assays further evaluation of nematode infection and calculation of the number of eggs/root system and eggs/gram of fresh root can be performed. For the tomato assays, individual roots are weighed and eggs extracted as described for inoculum preparation (section 3). When processing large number of plant samples, individual root systems could be macerated using a blender for egg extraction. The number of eggs should be counted in at least three aliquots and the eggs/gram of fresh root system calculated. For the pouch assays, the root system is pulled off the paper insert, weighed, and eggs extracted.

<table border="1">
	<tbody>
		<tr>
			<td>Compound </td>
			<td>Concentration </td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Ca(NO3)2 &bull; 4 H2O</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgSO4 &bull; 7H2O</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Hamp-ene Fe EDTA 13.0%</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>H3BO3</td>
			<td>46 &mu;M</td>
		</tr>
		<tr>
			<td>MnCl2 &bull; 4H2O</td>
			<td>9 &mu;M</td>
		</tr>
		<tr>
			<td>ZnSO4 &bull; 7H2O</td>
			<td>0.00076 &mu;M</td>
		</tr>
		<tr>
			<td>CuSO4 &bull; 5H2O</td>
			<td>0.00032 &mu;M</td>
		</tr>
		<tr>
			<td>(NH4)6Mo7O24 &bull; 4H2O</td>
			<td>0.00016 &mu;M</td>
		</tr>
	</tbody>
</table>
Table 1. Reagents for Hoagland's solution.
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent </td>
			<td>Company </td>
			<td>Catalogue number </td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Erioglaucine</td>
			<td>Fisher</td>
			<td>AC22973-0250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Miracle-Gro for tomato</td>
			<td>Scotts Miracle-Gro</td>
			<td>&nbsp;</td>
			<td>18-18-21</td>
		</tr>
		<tr>
			<td>Osmocote</td>
			<td>Scotts Miracle-Gro</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Seedling growth pouches</td>
			<td>Mega International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sieve 25 &mu;m</td>
			<td>H &amp; C Sieving Systems</td>
			<td>3886</td>
			<td>US standard #500</td>
		</tr>
		<tr>
			<td>Sieve 90 &mu;m</td>
			<td>H &amp; C Sieving Systems</td>
			<td>3880</td>
			<td>US standard #170</td>
		</tr>
		<tr>
			<td>Sieve 425 &mu;m</td>
			<td>H &amp; C Sieving Systems</td>
			<td>3871</td>
			<td>US standard #40</td>
		</tr>
		<tr>
			<td>Sunshine mix</td>
			<td>Sun Gro Horticulture Canada</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 2. Table of specific reagents and equipment.

high and low throughput screens with root-knot nematodes meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

Watch this Scientific Journal Video about High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp. at JoVE.com

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

high-low-throughput-screens-with-root-knot-nematodes-meloidogyne

Research

JoVE Journal

Immunology and Infection

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

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 ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are affected both by the doubling time of the microorganism and by any delay in growth upon transfer from one condition to another, the lag. The ScanLag method enables the characterization of these two independent properties at the level of colonies originating each from a single cell, generating a two-dimensional distribution of the lag time and of the growth time. In ScanLag, measurement of the time it takes for colonies on conventional nutrient agar plates to be detected is automated on an array of commercial scanners controlled by an in house application. Petri dishes are placed on the scanners, and the application acquires images periodically. Automated analysis of colony growth is then done by an application that returns the appearance time and growth rate of each colony. Other parameters, such as the shape, texture and color of the colony, can be extracted for multidimensional mapping of sub-populations of cells. Finally, the method enables the retrieval of rare variants with specific growth phenotypes for further characterization. The technique could be applied in bacteriology for the identification of long lag that can cause persistence to antibiotics, as well as a general low cost technique for phenotypic screens.

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...