1. Peptide Serial Dilution
<ol>
	<li>Prepare assay buffer: Hanks buffered salt solution (HBSS) supplemented with 25 mM HEPES, 0.1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), pH 7.4.</li>
	<li>Use a bulk reagent dispenser to systematically add 5 &#181;l of assay buffer to each well of five 384-well low volume assay plates.
	<ol>
		<li>Use internal software to create a dispensing program for 5 &#181;l volume addition to every well of a 384-well plate as per manufacturer&#39;s instructions.</li>
		<li>Immerse dispensing cassette tubing in assay buffer and prime fluid.</li>
		<li>Place 384-well low volume assay plate on plate carrier.</li>
		<li>Press start.</li>
	</ol>
	</li>
	<li>Dilute all peptide samples, regardless of storage vehicle, into assay buffer to produce a 100x peptide stock. 
	NOTE: Peptides requiring screening may be provided in Phosphate-buffered saline (PBS) or Dimethyl sulfoxide (DMSO) as is deemed appropriate (e.g., DMSO will have a deleterious effect on peptides with secondary modifications such as PEGylation).</li>
	<li>Dispense 25 &#181;l 100x peptide stocks into columns 1-5 of an acoustically qualified 384-well polypropylene microplate. This plate is designated &#39;source plate A&#39;. Ensure that source plates, but not destination plates, are flat bottomed and conform to specific acoustic tolerances as defined by the manufacturer of acoustic instruments.</li>
	<li>Dispense 25 &#181;l 100x reference control into wells A23 and A24 of source plate A.</li>
	<li>Dispense 10 &#181;l assay buffer into columns 11-15 and 30 &#181;l assay buffer into columns 21-22 of source plate A.</li>
	<li>Dispense 10 &#181;l assay buffer into columns 6-10 and columns 16-20 of a second acoustically qualified 384-well polypropylene microplate. This plate is designated &#39;source plate B&#39;.</li>
	<li>Centrifuge source plates A and B at 300 x g for 1 min. Include an appropriate balance plate.</li>
	<li>Use acoustic fluid dispensers integrated into an automated robotic system to prepare three sequential 1:100 intermediate dilutions (in assay buffer) of the 100x peptide stocks from column 1 in source A. 
	NOTE: Programming requires plate reformatting and dose response software (as provided by the manufacturer of the acoustic fluid dispenser) to allow three acoustic transfers between source A and source B (see Figure 1 for plate layouts). Step 1.9 details specifically the use of fluid dispensers used in this laboratory under automated robotic control (see Materials Table):
	<ol>
		<li>Load source plates and assay plates into plate hotel section of robotics.</li>
		<li>Open automated robotic software and load plate reformatting and dose response program expansion protocols. Click &#39;Run&#39;. 
		NOTE: Automation instructs an acoustic fluid dispenser to transfer 250 nl from columns 1-5 of source plate A into columns 6-10 of source plate B, and a second acoustic fluid dispenser capable of handling larger fluid volumes to backfill with 15 &#181;l assay buffer to mix. Automation then transfers source plate B to integrated centrifuge and centrifuges at 300 x g for 1 min (an appropriate balance plate is included for all centrifugation steps). Source plate B is returned to the acoustic fluid dispenser for the transfer of 250 nl from columns 6-10 of source plate B into columns 11-15 of source plate A and backfill with 15 &#181;l assay buffer to mix. Automation transfers source plate A to integrated centrifuge and centrifuges at 300 x g for 1 min. Source plate A is then returned to the acoustic fluid dispenser for the transfer of 250 nl from columns 11-15 of source plate A into columns 16-20 of source plate B and backfill with 15 &#181;l assay buffer to mix. Automation then transfers source plate B to integrated centrifuge and centrifuges at 300 x g for 1 min. 
		NOTE: Finally the dose response protocol directs acoustic dispensing to transfer the required volume from each of the 4 serially diluted source plate wells (in both source plate A and source plate B) to construct a full 11-point curve in duplicate in assay plates (pre-filled with 5 &#181;l assay buffer in step 1.2 above). Automation transfers assay plate to integrated centrifuge and centrifuges at 300 x g for 1 min.</li>
	</ol>
	</li>
</ol>
2. Cell Preparation
<ol>
	<li>Thaw cryopreserved Chinese hamster ovary (CHO) cells expressing the target mouse GLP-1 receptor rapidly in a 37 &#186;C water bath and resuspend in 20 ml assay buffer.</li>
	<li>Centrifuge cell suspension for 5 min at 200 x g at room temperature (RT). Include an appropriate balance.</li>
	<li>Discard supernatant and resuspend cell pellet in 10 ml assay buffer.</li>
	<li>Dilute cell stock 1:1 in Trypan blue and determine viable cell density using an automated cell counter. Resuspend cells in assay buffer at 1.6 x 106 cells per ml (equivalent to 8,000 cells per well of assay plates).</li>
	<li>Use a bulk reagent dispenser to add 5 &#181;l of cell suspension to each well of the assay plates (containing serially diluted peptides in 5 &#181;l assay buffer) and incubate at RT for 30 min.</li>
</ol>
3. HTRF cAMP Detection Assay
<ol>
	<li>Bring HTRF cAMP assay kit to RT for 30 min prior to use.</li>
	<li>Prepare each HTRF reagent (cryptate and d2) separately at 1:20 dilution in lysis buffer (proprietary formulation, provided by manufacturer of cAMP detection assay). 
	NOTE: CAUTION: Lysis buffer contains potassium fluoride (KF) which is toxic and a teratogen13. For disposal, assay plates containing KF should be sealed and incinerated, and any liquid waste must be diluted to &#60;1 mmol/L with water prior to disposal down the sink.</li>
	<li>Use a bulk reagent dispenser to add 5 &#181;l cryptate reagent to all wells of the assay plates.</li>
	<li>Use a bulk reagent dispenser to add 5 &#181;l d2 reagent to columns 1-22 of the assay plates.</li>
	<li>Immediately manually pipette 5 &#181;l lysis buffer into wells A23-D24 of the assay plates to give non-specific binding (NSB) control wells, and 5 &#181;l of d2 reagent into wells E23-P24.</li>
	<li>Centrifuge the assay plates for 1 min at 200 x g at RT to mix the wells.</li>
	<li>Cover the assay plates to minimize evaporation and photo-bleaching and incubate at RT for 1 hr.</li>
	<li>Measure fluorescence resonance energy transfer (FRET) signal using excitation at 320 nm and emission at 620 nm and 665 nm using a plate reader.</li>
</ol>
4. Data Analysis
<ol>
	<li>Use mean NSB to subtract background from all wells.</li>
	<li>Calculate %Delta F from the 665 nm/620 nm ratio as follows: 
	Ratio = (A665nm/A620nm) x 104 
	Delta F = ((Sample Ratio - RatioNSB)/RatioNSB)) x 100 
	where RatioNSB = wells with no d2 reagent.</li>
	<li>Calculate % activation between unstimulated cells and cells stimulated with maximum GLP-1 ligand as follows: 
	% activation = (%Delta Fsample - %DeltaFunstimulated cells)/(%DeltaFGLP-1 stimulated cells - %DeltaF unstimulated cells) x 100</li>
	<li>Analyze concentration-response curves via 4-parameter logistical analysis, and graph as % activation as defined by reference control1.</li>
</ol>

<ol>
	<li>Naylor, J., Rossi, A., Hornigold, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+Dispensing+Preserves+the+Potency+of+Therapeutic+Peptides+throughout+the+Entire+Drug+Discovery+Workflow.">Acoustic Dispensing Preserves the Potency of Therapeutic Peptides throughout the Entire Drug Discovery Workflow.</a> J.Lab.Autom. 21 (1), 90-96 (2016).</li><li>Campbell, J. E., Drucker, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology,+physiology,+and+mechanisms+of+incretin+hormone+action.">Pharmacology, physiology, and mechanisms of incretin hormone action.</a> Cell.Metab. 17 (6), 819-837 (2013).</li><li>Hui, H., Farilla, L., Merkel, P., Perfetti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+short+half-life+of+glucagon-like+peptide-1+in+plasma+does+not+reflect+its+long-lasting+beneficial+effects.">The short half-life of glucagon-like peptide-1 in plasma does not reflect its long-lasting beneficial effects.</a> Eur.J.Endocrinol. 146 (6), 863-869 (2002).</li><li>Harris, D., Olechno, J., Datwani, S., Ellson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient,+contact-free+volume+transfers+minimize+compound+loss+in+dose-response+experiments.">Gradient, contact-free volume transfers minimize compound loss in dose-response experiments.</a> J.Biomol.Screen. 15 (1), 86-94 (2010).</li><li>Ekins, S., Olechno, J., Williams, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispensing+Processes+Impact+Apparent+Biological+Activity+as+Determined+by+Computational+and+Statistical+Analyses.">Dispensing Processes Impact Apparent Biological Activity as Determined by Computational and Statistical Analyses.</a> PLoS ONE. 8 (5), 62325 (2013).</li><li>Goebel-Stengel, M., Stengel, A., Tache, Y., Reeve, 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=The+importance+of+using+the+optimal+plasticware+and+glassware+in+studies+involving+peptides.">The importance of using the optimal plasticware and glassware in studies involving peptides.</a> Anal.Biochem. 414 (1), 38-46 (2011).</li><li>McDonald, G. 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=Bioactive+Contaminants+Leach+from+Disposable+Laboratory+Plasticware.">Bioactive Contaminants Leach from Disposable Laboratory Plasticware.</a> Science. 322 (5903), 917 (2008).</li><li>Belaiche, C., Holt, A., Saada, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonylphenol+ethoxylate+plastic+additives+inhibit+mitochondrial+respiratory+chain+complex+I.">Nonylphenol ethoxylate plastic additives inhibit mitochondrial respiratory chain complex I.</a> Clin Chem. 55 (10), 1883-1884 (2009).</li><li>Sackmann, E. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achieving+accurate+compound+concentration+in+cell-based+screening:+validation+of+acoustic+droplet+ejection+technology.">Achieving accurate compound concentration in cell-based screening: validation of acoustic droplet ejection technology.</a> J.Biomol.Screen. 14 (5), 452-459 (2009).</li><li>Turmel, M., Itkin, Z., Liu, D., Nie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Innovative+Way+to+Create+Assay+Ready+Plates+for+Concentration+Response+Testing+Using+Acoustic+Technology.">An Innovative Way to Create Assay Ready Plates for Concentration Response Testing Using Acoustic Technology.</a> J.Lab.Autom. 15 (4), 297-305 (2010).</li><li>Butler, 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=Use+of+the+site-specific+retargeting+jump-in+platform+cell+line+to+support+biologic+drug+discovery.">Use of the site-specific retargeting jump-in platform cell line to support biologic drug discovery.</a> J.Biomol.Screen. 20 (4), 528-535 (2015).</li><li>Panchal, S., Verma, R. 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Aided Mol.Des. 29 (12), 1073-1086 (2015).</li><li>Harris, D., Olechno, J., Datwani, S., Ellson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient,+Contact-Free+Volume+Transfers+Minimize+Compound+Loss+in+Dose-Response+Experiments.">Gradient, Contact-Free Volume Transfers Minimize Compound Loss in Dose-Response Experiments.</a> J.Biomol. Screen. 15 (1), 86-94 (2010).</li><li>Chan, G. K. Y., Wilson, S., Schmidt, S., Moffat, 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=Unlocking+the+potential+of+high-throughput+drug+combination+assays+using+acoustic+dispensing.">Unlocking the potential of high-throughput drug combination assays using acoustic dispensing.</a> J.Lab.Autom. 21 (1), 125-132 (2016).</li><li>Roberts, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+and+challenges+of+direct+acoustic+dosing+into+cell-based+assays.">Implementation and challenges of direct acoustic dosing into cell-based assays.</a> J.Lab.Autom. 21 (1), 76-89 (2016).</li></ol>

The authors have nothing to disclose.

This protocol describes the successful application of automated acoustic dispensing to serially dilute peptide samples over a concentration range of 3 x 106 requiring less than 1 &#181;l of sample. The major advantage of this method is to increase data quality through minimizing peptide adsorption to plasticware via reduced exposure of samples to experimental containers and plasticware (such as pipette tips) that are normally required for reagent transfer and mixing. While acoustic dispensing does not complete...

The GLP-1R is an established drug target in the treatment of type 2 diabetes2. The native peptide agonist for this receptor, GLP-1, has an in vivo half-life of 2-3 min3. The binding of GLP-1 to its G protein coupled target receptor results in the downstream production of the second messenger cAMP through native G protein coupling to the activation of adenylyl cyclase. Measurement of the accumulated cAMP provides a robust assay to monitor receptor activation and to screen for active GLP-1 analogues with preferred physicochemical properties. Such an assay requires the serial dilution of test samples to construct concentration-response curves, and this is particularly complicated when handing peptide samples. Potential errors from tip-based serial dilution preparation have been described previously1,4,5. Peptides will adsorb to plasticware, resulting in unreliable potency estimations. Peptide loss can be minimized through the inclusion of bovine serum albumin (BSA) in buffers and the use of siliconized plasticware, yet protein binding remains unpredictable. In particular, the variation in binding of GLP-1 to experimental containers has been described6. There is a further complication in that stabilization agents used in laboratory plasticware can leach from tips and microtiter plates into aqueous assay buffers and interfere with protein function7, 8. Therefore, methods to reduce exposure to plasticware are necessary to increase the accuracy of measurements.
Acoustic liquid dispensers focus a high-frequency acoustic signal onto the surface of a fluid sample, resulting in the ejection of precise nanoliter droplets into an adjacent assay plate9. The use of acoustic ejection is standard in the pharmaceutical industry for the preparation and screening of large synthetic compound libraries, and the technology has been well validated for small molecules10. To our knowledge, we are the first group to describe acoustic dispensing for the preparation of recombinant and synthetic peptides and we have previously reported the improved accuracy compared to conventional tip-based methods1.
This article describes the integration of the preparation of peptide serial and direct dilutions by non-contact acoustic transfer onto a fully automated plate handling robotics system. A number of methods encompassing acoustic transfer of samples have been described previously11. We utilize a two-step method to prepare intermediate stock concentrations and to serially dilute peptide analogues for the generation of the full dose-response curve. The prepared peptides are incubated with cells expressing the target mouse GLP-1R, and we use a commercially available homogenous time-resolved fluorescence (HTRF) assay to measure cAMP accumulation within these cells as a readout of peptide agonist activity. The assay is robust and amenable to a high-throughput 384-well format and routinely applied to both assay development and drug screening projects12.

<table><tbody><tr><td>Hanks&amp;rsquo; Balanced Salt solution</td><td>Sigma-Aldrich</td><td>H8264</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I7018</td><td>Prepared as a 0.5 M stock in DMSO</td></tr><tr><td>GLP-1 (7-36) amide</td><td>Bachem</td><td>H-6795</td><td>Prepared as a 1 mg/ml stock in PBS, referred to as &#039;100x reference control&#039;</td></tr><tr><td>Test peptides</td><td>Produced in-house at MedImmune</td><td></td><td>Supplied at various concentrations in DMSO or PBS as appropriate</td></tr><tr><td>100x peptide stock</td><td>Produced in-house at MedImmune</td><td></td><td>Test peptide diluted into assay buffer to 100x final required concentration</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250-061</td><td></td></tr><tr><td>Cedex XS Cell Analyzer</td><td>Innovatis</td><td></td><td></td></tr><tr><td>Corning 384 well plates, low volume</td><td>Sigma-Aldrich</td><td>4514</td><td></td></tr><tr><td>Echo Qualified 384-Well Polypropylene Microplate</td><td>Labcyte Inc.</td><td>P-05525</td><td></td></tr><tr><td>Echo Qualified Reservoir</td><td>Labcyte Inc.</td><td>ER-0055</td><td></td></tr><tr><td>Echo 550 Liquid Handler</td><td>Labcyte Inc.</td><td></td><td>Droplet transfer volumes in increments of 2.5 nl</td></tr><tr><td>Echo 525 Liquid Handler</td><td>Labcyte Inc.</td><td></td><td>Droplet transfer volumes in increments of 25 nl</td></tr><tr><td>ACell Benchtop Automation&amp;nbsp;</td><td>HighRes Biosolutions</td><td>MC522</td><td></td></tr><tr><td>Cellario Lab Automation Scheduling software for Life Science Robotics</td><td>HighRes Biosolutions</td><td></td><td></td></tr><tr><td>MultidropCombi Reagent Dispenser</td><td>ThermFisher Scientific</td><td>5840300</td><td>Referred to as &#039;bulk reagent dispenser&#039;</td></tr><tr><td>HTRF cAMP Dynamic 2 kit</td><td>Cisbio Bioassays</td><td>62AM4PEJ</td><td></td></tr><tr><td>EnVision Multilabel Reader</td><td>PerkinElmer</td><td></td><td></td></tr></tbody></table>

automated acoustic dispensing for the serial dilution of peptide agonists in potency determination assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

We routinely use a two-step method to dilute peptides via acoustic transfer. For the first step, an acoustic dispenser aligned with automation is used to create four stock peptide intermediate dilutions across two source plates (Figure 1a, b). For the second step, we use an acoustic dispenser to further dilute stock dilutions from source plates A and B to create an 11-point concentration range for each test peptide (Figure 1c). Each pepti...

Watch this Scientific Journal Video about Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays at JoVE.com

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

automated-acoustic-dispensing-for-serial-dilution-peptide-agonists

Research

JoVE Journal

Biochemistry

7.6K Views.  MedImmune. Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described. 

Video: Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Genetics

Reliable Mechanochemistry: Protocols for Reproducible Outcomes of Neat and Liquid Assisted Ball-mill Grinding Experiments

The equilibrium outcomes of ball mill grinding can dramatically change as a function of even tiny variations in the experimental conditions such as the presence of very small amounts of added solvent. To reproducibly and accurately capture this sensitivity, the experimentalist needs to carefully consider every single factor that can affect the ball mill grinding reaction under investigation, from ensuring the grinding jars are clean and dry before use, to accurately adding the stoichiometry of the starting materials, to validating that the delivery of solvent volume is accurate, to ensuring that the interaction between the solvent and the powder is well understood and, if necessary, a specific soaking time is added to the procedure. Preliminary kinetic studies are essential to determine the necessary milling time to achieve equilibrium. Only then can exquisite phase composition curves be obtained as a function of the solvent concentration under ball mill liquid assisted grinding (LAG). By using strict and careful procedures analogous to the ones here presented, such milling equilibrium curves can be obtained for virtually all milling systems. The system we use to demonstrate these procedures is a disulfide exchange reaction starting from the equimolar mixture of two homodimers to obtain at equilibrium quantitative heterodimer. The latter is formed by ball mill grinding as two different polymorphs, Form A and Form B. The ratio R = [Form B] / ([Form A] + [Form B]) at milling equilibrium depends on the nature and concentration of the solvent in the milling jar.

We present detailed procedures to produce experimental equilibrium curves of the phase composition as a function of solvent concentration in a solid state system under milling conditions.

The equilibrium outcomes of ball mill grinding can dramatically change as a function of even tiny variations in the experimental conditions such as the ...

Chemistry

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. ...

Immunology and Infection

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Bioengineering

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or lethality. However, the probability of a synthetic small molecule being bioactive is low; therefore, thousands of molecules must be tested in order to find those of interest. Liquid handling robotics systems are designed to handle large numbers of samples, increasing the speed with which a chemical library can be screened in addition to minimizing/standardizing error. To achieve a high-throughput forward chemical genetics screen of a library of 50,000 small molecules on Arabidopsis thaliana (Arabidopsis), protocols using a bench-top multichannel liquid handling robot were developed that require minimal technician involvement. With these protocols, 3,271 small molecules were discovered that caused visible phenotypic alterations. 1,563 compounds induced short roots, 1,148 compounds altered coloration, 383 compounds caused root hair and other, non-categorized, alterations, and 177 compounds inhibited germination.

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

A high throughput screen of synthetic small molecules was conducted on the model plant species, Arabidopsis thaliana. This protocol, developed for a liquid handling robot, increases the speed of forward chemical genetics screens, accelerating the discovery of novel small molecules affecting plant physiology.

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Biology

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Samenvatting

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