Name: high-throughput dna plasmid multiplexing and transfection using acoustic nanodispensing technology
Uploaded: 2019-08-08T13:00:00
Description: Watch this Scientific Journal Video about High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology at JoVE.com

The authors disclosed a receipt of the following financial support for the research, authorship, and/or publication of this article: Inserm, Lille University, Lille Pasteur Institute, Conseil R&#233;gional du Nord, and PRIM-HCV1 and 2 (P&#244;le de Recherche Interdisciplinaire sur le M&#233;dicament), Agence Nationale de la Recherche (ANR-10-EQPX-04-01), the Feder (12001407 (D-AL) Equipex Imaginex BioMed) and the European Community (ERC-STG INTRACELLTB n&#176; 260901). The authors wish to thank Dr. S. Moureu, Dr. B. Villemagne, Dr. R. Ferru-Cl&#233;ment, and Dr. H. Groult for their critical review and corrections of the manuscript.

1. Advance preparations
<ol>
	<li>Preparation of the peristaltic liquid handler programs 
	NOTE: For the diluent and cell dispensing steps of the protocol, a dedicated program must be prepared, taking into account the height of the dispensing head to the plate used and the step intent.
	<ol>
		<li>For the 1 &#181;L diluent dispense step, mount a 1 &#181;L cassette and prepare a program with the settings as described in steps 1.1.1.1 and 1.1.1.2.
		<ol>
			<li>Adjust the flow rate parameter to <str...

<ol>
	<li>Colin, B., Deprez, B., Couturier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+DNA+Plasmid+Transfection+Using+Acoustic+Droplet+Ejection+Technology.">High-Throughput DNA Plasmid Transfection Using Acoustic Droplet Ejection Technology.</a> SLAS Discovery: Advancing Life Sciences R &#38; D. , 2472555218803064 (2018).</li><li>Mirus Bio. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optimising Transfection Performance. , (2019).</li><li>Thermo Fisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Factors Influencing Transfection Efficiency | Thermo Fisher Scientific - FR. , (2019).</li><li>Boussif, O., 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+versatile+vector+for+gene+and+oligonucleotide+transfer+into+cells+in+culture+and+in+vivo:+polyethylenimine.">A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (16), 7297-7301 (1995).</li><li>Figueroa, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanistic+investigation+exploring+the+differential+transfection+efficiencies+between+the+easy-to-transfect+SK-BR3+and+difficult-to-transfect+CT26+cell+lines.">A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines.</a> Journal of Nanobiotechnology. 15 (1), 36 (2017).</li><li>Kirchenbuechler, I., Kirchenbuechler, D., Elbaum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+cationic+lipid-based+transfection+and+cell+division.">Correlation between cationic lipid-based transfection and cell division.</a> Experimental Cell Research. 345 (1), 1-5 (2016).</li><li>Zhang, Z., Qiu, S., Zhang, X., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+DNA+electroporation+for+primary+human+T+cell+engineering.">Optimized DNA electroporation for primary human T cell engineering.</a> BMC Biotechnology. 18 (1), 4 (2018).</li><li>Cao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+activity+and+the+mechanism+of+pDNA-complexes+based+on+the+hybrid+of+low-generation+PAMAM+and+branched+PEI-1.8k.">Transfection activity and the mechanism of pDNA-complexes based on the hybrid of low-generation PAMAM and branched PEI-1.8k.</a> Molecular bioSystems. 9 (12), 3175-3186 (2013).</li><li>Bos, A. B., 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=Development+of+a+semi-automated+high+throughput+transient+transfection+system.">Development of a semi-automated high throughput transient transfection system.</a> Journal of Biotechnology. 180, 10-16 (2014).</li><li>Colosimo, 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=Transfer+and+expression+of+foreign+genes+in+mammalian+cells.">Transfer and expression of foreign genes in mammalian cells.</a> BioTechniques. 29 (2), 314-318 (2000).</li><li>Villa-Diaz, L. G., Garcia-Perez, J. L., Krebsbach, 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=Enhanced+transfection+efficiency+of+human+embryonic+stem+cells+by+the+incorporation+of+DNA+liposomes+in+extracellular+matrix.">Enhanced transfection efficiency of human embryonic stem cells by the incorporation of DNA liposomes in extracellular matrix.</a> Stem Cells and Development. 19 (12), 1949-1957 (2010).</li><li>Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Reverse transfection method. , WO2001020015A1 (2001).</li><li>Raymond, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+polyethylenimine-mediated+transfection+process+for+large-scale+and+high-throughput+applications.">A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications.</a> Methods (San Diego, CA). 55 (1), 44-51 (2011).</li><li>Junquera, E., Aicart, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+gene+therapy+to+deliver+nucleic+acids+with+multivalent+cationic+vectors.">Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors.</a> Advances in Colloid and Interface Science. 233, 161-175 (2016).</li><li>Woodruff, K., Maerkl, S. 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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> Journal of Biomolecular Screening. 14 (5), 452-459 (2009).</li><li>Sackmann, E. 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=Technologies+That+Enable+Accurate+and+Precise+Nano-+to+Milliliter-Scale+Liquid+Dispensing+of+Aqueous+Reagents+Using+Acoustic+Droplet+Ejection.">Technologies That Enable Accurate and Precise Nano- to Milliliter-Scale Liquid Dispensing of Aqueous Reagents Using Acoustic Droplet Ejection.</a> Journal of Laboratory Automation. 21 (1), 166-177 (2016).</li><li>Day, R. N., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+protein+palette:+tools+for+cellular+imaging.">The fluorescent protein palette: tools for cellular imaging.</a> Chemical Society Reviews. 38 (10), 2887-2921 (2009).</li><li>Zielinski, D., Gordon, A., Zaks, B. L., Erlich, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPipet:+sample+handling+using+a+tablet.">iPipet: sample handling using a tablet.</a> Nature Methods. 11 (8), 784-785 (2014).</li><li>Brunner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+dependence+of+gene+transfer+by+lipoplex,+polyplex+and+recombinant+adenovirus.">Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus.</a> Gene Therapy. 7 (5), 401-407 (2000).</li><li>Nii, 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=Single-Cell-State+Culture+of+Human+Pluripotent+Stem+Cells+Increases+Transfection+Efficiency.">Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency.</a> BioResearch Open Access. 5 (1), 127-136 (2016).</li><li>Noonan, D. J., Henry, K., Twaroski, M. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+dry+DNA+storage+and+shipment+at+room+temperature.">Protocols for dry DNA storage and shipment at room temperature.</a> Molecular Ecology Resources. 13 (5), 890-898 (2013).</li><li>Lesnick, J., Lejeune-Dodge, A., Ruppert, N., Jarman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High-Precision Cell Dispensing with the Labcyte Echo&#174; Liquid Handler. , (2017).</li><li>Yang, X., 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+public+genome-scale+lentiviral+expression+library+of+human+ORFs.">A public genome-scale lentiviral expression library of human ORFs.</a> Nature Methods. 8 (8), 659-661 (2011).</li><li>Peng, J., Zhou, Y., Zhu, S., Wei, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screens+in+mammalian+cells+using+the+CRISPR-Cas9+system.">High-throughput screens in mammalian cells using the CRISPR-Cas9 system.</a> The FEBS journal. 282 (11), 2089-2096 (2015).</li></ol>

The establishment and optimization of an accurate high-throughput transfection method for a given cell line require scientists to follow some key parameters described in this section. We strongly encourage starting with the recommended values throughout the protocol as these settings optimized for HeLa cells also proved to be efficient for HEK cells. However, as the best parameters may depend on the cell lines and transfection reagents, optimal conditions can be defined by&#160;varying the cell number, diluent volume, to...

The method presented here describes in detail how to perform DNA plasmid multiplexing and transfection in mammalian cells at high throughput using an acoustic-based liquid nanodispenser in a 384-well plate, even for nonspecialists in the field. This recently published method1 allows performing as much as 384 independent plasmid DNA multiplexing and transfection conditions in one experiment, in less than 1 h. Single or cotransfection experiments were successful, reaching a near 100% cotransfection within the transfected cells population. This protocol makes transfection easier because most of the tedious, time-consuming, and error-prone steps are now software driven (see Figure 1 for a general overview). Further efforts have been made to develop dedicated tools to enhance the ease of use while avoiding human errors during the overall process and to promote successful transfection even for nonspecialists in the field. The described protocol includes a &#34;user-friendly&#34; macro spreadsheet that we developed in order to manage 384 independent transfection conditions with multiplexing possibilities of up to four plasmids in each well. The macro automatically generates templates of the source plate(s) to load the expected DNA plasmid volume from starting stock solutions and the files required to drive the nanodispenser software upon the experimental design that has been inputted. As the manual dispensing of DNA in a 384-well source plate is tedious and error-prone, we also developed a dedicated tablet-based application to guide the user while dispensing DNA solution according to the template.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59570/59570fig1v3.jpg" /> 
Figure 1: Experimental workflow. Schematic representation of the optimal automated high-throughput reverse transfection protocol (from experimental design to custom biological assay). Manual steps are indicated by the hand symbol and the approximate time for each step is written in a red box. <a href="https://www.jove.com/files/ftp_upload/59570/59570fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Many cell-based experiments start with plasmid DNA transfection, and even if many dedicated reagents have been and are still being developed to enhance transfection efficiency and/or ease the procedure, much remains to be done2,3,4. DNA plasmid cell transfection involves several steps to reach high efficiency, such as an initial complex uptake, endosomal escape, and cytoplasmic transport to the nucleus5,6. In addition to calcium precipitation or physical techniques such as electroporation or microinjection using dedicated devices7, modern chemical methods have focused on enhancing DNA cell delivery while lowering the cell cytoxicity8,9. The use of lipids or cationic polymers forming liposome-like complexes and, more recently, nonliposomal polymeric chemistry systems has made transfection easier and more efficient10. Despite these developments, cell transfection still requires specific skills to be accurately performed as most of these physical or chemical transfection protocols require scientists to manually prepare each DNA transfection reaction condition, thus impairing the throughput. To circumvent this problem, reverse transfection protocols have been developed using chemical transfection reagents11,12,13, enabling the user to test or combine several plasmids in a faster way. In these protocols, nucleic acid complexes with transfection reagents are formed before seeding the cells on the complexes. However, these reverse protocols are still limited by the manual handling of DNA solutions and by the combination of each of the independent conditions. Although it is feasible to perform them in a 96-well plate format, the DNA preparation and dispenses will be tedious, and there likely will be mistakes. When different amounts of several DNA plasmids are required and multiplexed with each other, cell transfection becomes even harder to achieve and more time-consuming, and human errors become quite inevitable. Scaling up to the 384-well plate format in a reverse transfection approach, in spite of few multiplexed DNA transfection conditions, becomes an impossible challenge due to the following reasons. i) The DNA amounts, transfection reagent, or reaction mixture volumes to manage are lower than 1 &#181;L for each well. ii) The multiplexing of plasmids for 384 independent conditions becomes extremely complicated. The delivery in each of the 384 wells is also iii) highly time-consuming and iv) error-prone. Indeed, dispensing the right solution in the expected wells is hard to manage because the low volumes already dispensed do not allow visual monitoring between the empty and already filled wells. v) Finally, there is a high risk of drying the mixture by evaporation before the cells are added due to the time needed to perform the necessary dispensing steps. In summary, the limiting factor to set up high-throughput DNA plasmid transfection assays appears to be the miniaturization of the assay, which implies low-volume multiplexing and managing that cannot be handled manually anymore but are also hardly achievable in a reliable way by classical peristatic liquid handlers.
As a proof of difficulty to automatize such assays and gain high-throughput, only a few attempts to automate transfection have been published so far: a 96-well plate format using a commercial liquid handling device and calcium phosphate precipitation14 and, more recently, a lipoplex reagent, and a microfluidic chip enabling 280 independent transfections15 but requiring specialized skills in this field. Another method, acoustophoresis, allowing liquid levitation and leading to fluid manipulation and mixing, was used to perform DNA transfection in 24- to 96-well plate formats16. Although feasible, this approach suffers from an extremely low throughput as the mixing of cells with DNA transfection mixture requires a 60 s incubation for every single point before seeding. This implies a duration of at least 96 min for a complete 96-well plate. Furthermore, this protocol is far away from being amenable to the overall biologists&#39; audience as this work was done with an in-house designed and manufactured device which is currently not available on the market. On the contrary, in the last few years, an easy to use software-driven acoustic-based dispensing technology has emerged with nanovolume dispenser devices. Using focused acoustic energy, these devices allow the tightly controlled ejection of small liquid volumes from 2.5 nL to 500 nL from a source plate to a destination one17. This technology, called acoustic droplet ejection (ADE), has numerous advantages: it is fully automated, contactless, tipless, accurate, precise, and highly reproducible, and it has a high throughput18. First devoted to delivering dimethyl sulfoxide (DMSO) solutions, settings have been enhanced to dispense aqueous buffers19. Acoustic nanodispensers, then, seem suitable for reverse cell transfection protocols and could circumvent most of the above-mentioned manual limitations. As no plasmid transfection attempts were previously described using this technology, we recently evaluated the suitability of an acoustic-based dispensing system to perform reverse cell transfection.
Taking advantage of the nanodispenser throughput and ease of use, we optimized a reverse transfection protocol for HeLa cells by cross-testing several parameters that can influence DNA transfection on a 384-well, single plate, namely, the total DNA amount and source DNA starting concentration, diluent volume, transfection reagent, and number of spread cells. The developed protocol circumvents the above-described manual limitations of cell transfection and presents several advantages over other automated transfection attempts. First, it is miniaturized, thus allowing for cost-effective transfection reagent by saving DNA plasmid preparations and transfection reagent. Secondly, it is much more high-throughput and reproducible than the manual protocol (even for beginners), as transfection of an entire 384-well plate can be achieved in less than 1 h. Finally, it is software-driven, allowing the control of the dispensed DNA amount and the multiplexing of several plasmids. Indeed, thanks to the nanodispenser software (Table of Materials), the user can elaborate a study plan to control the volumes to be dispensed from a defined source well plate to a destination one.
The protocol presented here is mainly intended for those who have access to a nanodispenser and would like to set up transfection experiments at high throughput, but also for those who want to rapidly optimize their transfection parameters for a given cell type by applying this protocol to cross-test several parameters at high throughput. Indeed, we have shown that optimized parameters identified with this nanoscale protocol can be transposed to larger-scale and manual transfection experiments. Finally, as the transfection reagent used in the present protocol allows DNA or siRNA transfection according to the manufacturer, the protocol is also of interest to those aiming at performing array approaches for gene overexpression or knockdown. The destination plates prefilled with DNA can be conserved up to 7 days before use in a transfection assay without loss of efficacy, which is another advantage of the following protocol for this kind of application.

<table border="0" cellpadding="0" cellspacing="0" style="width:1226px;" width="1224">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">384LDV Microplate</td>
			<td style="width:363px;">Labcyte</td>
			<td style="width:202px;">LP-0200</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">384-well Microplate &#956;Clear Black</td>
			<td style="width:363px;">Greiner</td>
			<td style="width:202px;">781906</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Ampicilin</td>
			<td style="width:363px;">Sigma</td>
			<td style="width:202px;">A9393-5G</td>
			<td style="width:291px;">Selection antibiotic for bacteria transformed with ampicilin expressing vector</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Android Tablet</td>
			<td style="width:363px;">Samsung</td>
			<td style="width:202px;">Galaxy Note 8</td>
			<td style="width:291px;">used to guide the user while the source plate manual dispense</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Aniospray Surf 29</td>
			<td style="width:363px;">Anios</td>
			<td style="width:202px;">2421073</td>
			<td style="width:291px;">disinfectant to clean the MicroFlo head</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Columbus software</td>
			<td style="width:363px;">Perkin Elmer</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">image analysis software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX Supplement, pyruvate</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">10566032</td>
			<td style="width:291px;">cell culture medium</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:371px;">Echo Cherry Pick 1.5.3 software</td>
			<td style="width:363px;">Labcyte</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">Software enabling ADE-based dispenses by the Echo550 device from a *.csv file; nanodispenser software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Echo550</td>
			<td style="width:363px;">Labcyte</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">ADE-based dispenser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Fetal Bovine Serum</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">16000044</td>
			<td style="width:291px;">to add in cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:363px;">Sigma-Aldrich</td>
			<td style="width:202px;">HT501128-4L</td>
			<td style="width:291px;">to fix cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">HeLa cells</td>
			<td style="width:363px;">ATCC</td>
			<td style="width:202px;">HeLa (ATCC&#174; CCL-2&#8482;)</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">H3570</td>
			<td style="width:291px;">10 mg/mL Solution in Water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">INCell Analyzer 6000</td>
			<td style="width:363px;">GE Healthcare</td>
			<td style="width:202px;">29043323</td>
			<td style="width:291px;">automated laser-based confocal imaging platform</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:371px;">LB medium</td>
			<td style="width:363px;">Thermoischer Scientific 
			LB Broth Base (Lennox L Broth Base)&#174;, powder</td>
			<td style="width:202px;">12780052</td>
			<td style="width:291px;">culture medium for bacteria growth</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Lysis Buffer (A2)&#160;</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740912.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">MicroFlo 10&#181;L cassette</td>
			<td style="width:363px;">Biotek Instruments Inc</td>
			<td style="width:202px;">7170013</td>
			<td style="width:291px;">to use with the Microflo Dispenser</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">MicroFlo 1&#956;L cassette</td>
			<td style="width:363px;">Biotek Instruments Inc</td>
			<td style="width:202px;">7170012</td>
			<td style="width:291px;">to use with the Microflo Dispenser</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">MicroFlo Dispenser</td>
			<td style="width:363px;">Biotek Instruments Inc</td>
			<td style="width:202px;">7171000</td>
			<td style="width:291px;">peristaltic pump-based liquid handler device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microvolume spectrophotometer</td>
			<td style="width:363px;">Denovix</td>
			<td style="width:202px;">DS-11 Spectrophotometer</td>
			<td style="width:291px;">Measure the DNA concentration of samples</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:371px;">mVenus plasmid</td>
			<td style="width:363px;">mVenus cDNA was cloned by enzymatic restriction digestion and ligation in Age1/BsrG1 sites of the tdTomato-N1 plasmid</td>
			<td style="width:202px;"></td>
			<td style="width:291px;">Vector type: Mammalian Expression, Fusion Protein: mVenus</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Neutralization Buffer (A3)&#160;</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740913.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">NucleoSpin Plasmid kit</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740588.50</td>
			<td style="width:291px;">used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Optimal-Modified Eagle Medium (Opti-MEM) Medium</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">31985070</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">optional Wash bufferWash Buffer (A4)</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740914.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:50px;">orbital shaker&#160;</td>
			<td style="width:363px;">incubated large capacity shaker</td>
			<td style="width:202px;">444-7084</td>
			<td style="width:291px;">Used to grow bacteria under gentle agitation and 37&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Penicillin-Streptomycin</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">15140122</td>
			<td style="width:291px;">10,000 U/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Phosphate-Buffered Saline</td>
			<td style="width:363px;">Thermo Fisher Scientific</td>
			<td style="width:202px;">10010001</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Plasmid mini-columns</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740499.250</td>
			<td style="width:291px;">Silica membrane mini-column to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Resuspension Buffer (A1)&#160;</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740911.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">RNAse A</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740505</td>
			<td style="width:291px;">Enzyme from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">tdTomato-N1 plasmid</td>
			<td style="width:363px;">Addgene</td>
			<td style="width:202px;">Plasmid #54642</td>
			<td style="width:291px;">Vector type: Mammalian Expression, Fusion Protein: tdTomato&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">TransIT-X2 Dynamic Delivery System</td>
			<td style="width:363px;">Mirus Bio</td>
			<td style="width:202px;">MIR 6000</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:371px;">Wash Buffer (AW)</td>
			<td style="width:363px;">Macherey-Nagel</td>
			<td style="width:202px;">740916.1</td>
			<td style="width:291px;">Buffer from the NucleoSpin Plasmid kit used to prepare plasmid from bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">3D printer</td>
			<td style="width:363px;">Creality</td>
			<td style="width:202px;">CR10S</td>
			<td style="width:291px;">used to print the plate adapter</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:371px;">Blender Software&#160;</td>
			<td style="width:363px;">&#160;https://www.blender.org/ 
			Free software under GNU General Public License (GPL).</td>
			<td style="width:202px;">version 2.79b</td>
			<td style="width:291px;">used to design the plate adapter</td>
		</tr>
	</tbody>
</table>

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.

fIn order to determine if the ADE technology could be used for an automated reverse transfection protocol, we monitored cell transfection efficiency by fluorescence microscopy, using a red fluorescent tdTomato expressing plasmid. First aiming at determining the best transfection parameters, different diluent volumes and total amounts of DNA were cross-tested. Diluent volume was used to allow the DNA droplets, once dispensed, to spread all over the wells to circumvent inhomogeneous transfe...

Watch this Scientific Journal Video about High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology at JoVE.com

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

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 ...

The NanoDispenser based transfection protocol on developed to describe in this video represents the first user-friendly, high-throughput transfection method allowing even beginners in the field to be successful. This is easy on high-throughput technique allows transfection of cell with 384 independent condition. As of now, the method is low cost due to the nano volume of reagent needed.To define the best transfection parameters for all the cell type, we suggest keeping the source DNA concentration constant, and using varying amounts of transfected DNA, transfection reagent, and number of cells. For dispensing the 40 microliter cell suspension, mount a 10 microliter cassette on the peristaltic liquid handler device, and prepare an appropriate program. First, adjust the flow rate parameter to low to dispense cells with a low speed to avoid promoting potential damage to the cells by sheer stress and high impact on the bottom of the wells.Then, adjust the dispensation height to 11.43 millimeters. The height must be high enough to lower the cell impact on the bottom of the wells during the dispensing process, but low enough to avoid retention of the droplets on the dispensing head. Next, adjust the plate clear height to 16 millimeters to allow free displacement of the dispensing head over the plate after dispensing each row.Visually control the proper settings of the peristaltic liquid handler head height. Make sure no drops are retained on the dispensing tips while dispensing. Also verify that the head is high enough to allow displacement of the head after dispensing each row.To generate picklists to drive the ADE-dispensation, a user-friendly spreadsheet macro was developed to manage DNA amounts and mix up to four plasmids in a 384 well plate format. Optimal volumes are pre-filled in some cells of the spreadsheet, but can be changed. In the pink fields, the transfection reagent, or TR mixture value, is set to 500 nanoliters.The minimal volume value in the source plate wells is set to four microliters, and the maximal volume in the source plate wells to 11.25 microliters. Enter 100 nanograms per microliter DNA starting concentrations in the blue fields corresponding to the underlying DNA. Then, enter the desired DNA amount in the gray and green fields corresponding to the 384 wells of the plate.Enter the amounts and plasmid names and ensure the same spelling is used if the same plasmid has to be transfected in several wells. Click on generate picklists to verify the entered values, and if requested, correct the orange-filled cells indicating errors or volumes that cannot be handled by the NanoDispenser. If none are detected, the macro will generate the 384 well dispensing guide, the DNA picklist file, and the TR picklist file from data collected on the corresponding sheets.Print the template from the source plate sheet to visualize the wells to be filled before dispensing. Plasmid names and minimal fill volumes are indicated. Transfection reagent mixture volumes that will be filled in the following wells, are indicated as TR and highlighted in green.To prepare the DNA source plate, dilute the stored DNA plasmid to 100 nanograms per microliter using distilled water. Now, calibrate the 384 well grid to the plate dimensions. Open the 384 Well Pipetting Guide Application on a tablet.Place the source place on the grid on the lower screen. In the upper left calibration menu, click plus or minus to enhance or reduce the size of the grid and wells in order to adjust the green wells to the four corner wells of the plate. Using double sided tape, mount the 3D printed plate adapter on the screen to avoid source plate movements while dispensing.If needed, move the calibrated grid using the rotation arrows and up, down, right, left buttons to adjust the grid to the plate position. Once the grid and well sizes are properly calibrated and located, tick the lock calibration box. Click on file and open the 384-Wells-Pipetting-Guide.csv file. Follow the screen instructions to manually dispense the indicated volume of plasmid at the specified concentration into the white highlighted well that corresponds to the proper target destination of the expected plate. Use minus or plus arrows to go back or further in the DNA dispensing process.Stop dispensing upon reaching the first transfection reagent solution to load. Once the DNA dispensations are finished, remove the source plate from the adapter. If several source plates are to be filled, place a new source plate on the adapter and follow the dispensing instructions.Centrifuge the DNA filled source plates to ensure proper liquid leveling and to remove bubbles leading to inaccuracy in the ADE base transfers. Now, run a survey to control the manually dispensed volumes. On the NanoDispenser PC, run the NanoDispenser program.Go to the diagnostic tab, tick the source plate outbox, load the source plate on the plate holder and tick in to enter the plate. When prompted, select 384LDV_AQ_B2 to set the NanoDispenser to the aqueous buffer dispensing mode and press OK.Select survey in the miscellaneous menu. And click on launch.Select the prefilled wells to analyze and click on the go button. Verify that the measured volumes match the expected ones and ensure no wells have been loaded with volumes of more than 12 microliters. Prime the tubing with serum free medium by filling a new sterile vessel with 10 milliliters of pre-warmed serum free medium and submerging the tube organizer in it.Press the prime button of the peristaltic liquid handler for about 10 seconds. Make sure the tip is not clogged by visually inspecting the flow from the dispensing head. Place a sterile 384 well culture plate on the peristaltic liquid handler plate carrier, and remove its lid.Run the pre-calibrated program to dispense one microliter in each well of the 384 well plate. The dispensing time is approximately eight seconds. Then replace the lid of the 384 well plate.To set up ADE driven DNA dispensing, run the picklist software. Set the 384 wells sourced to 384LDV. Set the device to aqueous buffer dispensing mode by selecting 384LDV_AQ_B2.And the destination plate types to Greiner_384PS_781096. Un-tick optimized transfer throughput. Select pick list tab.Click on import. And select the DNA_Picklist_CSV file. Then, click on play and save the protocol.Click on simulate to perform a simulation of the program dispensations to make sure that the picklist matches the expected experimental design. Click on the run button and start the dispensing program. When asked, insert the requested source plate.And then the destination plate in the NanoDispenser. To set up the transfection reagent dispensation, work in a biosafety cabinet to dilute lipopolyplex transfection reagent in serum free medium to a one x final concentration and vortex the tube. Immediately dispense the TR mix according to the predefined source plate deigned by the macro and using the pre-calibrated 384 well pipetting guide application.Following TR dispensing, perform a survey to control the TR loaded volumes as done before for the loaded DNA. Select the transfection reaction mix prefilled wells to analyze. And click on the go button.Verify that the measured volumes match the expected ones. And ensure no wells have been loaded with volumes of more than 12 microliters. Now perform a reset of the DNA picklist in the picklist software.Verify that the device parameters are still set to aqueous buffers. Enter the correct source and destination plate types. On the pick list tab, click on reset to clear the sample list.Then click on import and choose the TR_Picklist. CSV file. Click on play.Save the protocol if prompted and perform a simulation of the program transfection reagent mixture dispensations to ensure proper design by clicking on the simulate button. As done previously, after clicking on the run button and starting the dispensing program place the source plate and then the destination plate in the NanoDipsenser as requested. To dispense the cells, fill a new sterile vessel with the prepared cell suspension and stir it to avoid sedimentation leading to inaccuracy and cell density.Insert the tube organizer in the solution. Then, press the prime button until the cell suspension starts to flush from the dispensing head. Make sure the tip is not clogged by visually inspecting the flow from the dispensing head while flushing and ensure each tube is loaded with cell suspension.Now, load the DNA and TR filled 384 well destination plate on the peristaltic liquid handler plate carrier and remove its lid. Run the pre-calibrated program to dispense 40 microliters of the cell suspension on the complete 384 well plate. The dispensing time is about 45 seconds.Finally, replace the lid of the 384 well plate. The transfection of HeLa cells using lipopolyplex reagent was successful. DNA amounts ranging from five to 30 nanograms showed the same efficiency and up to 90%cell transfection at one microliter diluent volume.In contrast, higher amounts resulted in an abrupt decrease in percentage of transfected cells. Various diluent volumes ranging from 15 nanoliters to four microliters were tested and one microliter was identified as the best condition. To further enhance the throughput of this protocol two efficient DNA storage methods were tested namely dry storing the plate or frozen storage.Both storage methods did not lead to significantly different results than freshly dispensed DNA solution stored for up to seven days. As plasma transfection usually employs at least two different plasmids, the DNA multiplexing ability of this protocol was examined using the best identified conditions. The previously used tdTomato red fluorescent protein expressing plasmid was modified to express mVenus a bright yellow fluorescent protein and both were then used in cotransfection attempts.Red or green fluorescent positive cell analysis showed the transfection efficiency to be about 80%However, nearly 100%of the red cells were also cotransfected with the mVenus expressing plasmid as can be seen in the representative software based image analysis. Once DNAs have been dispensed in the source plate. Perform a survey to ensure that the expected volumes have been loaded and do not exceed 12 microliters.While dispensing the transfection reagent in the source plate, try to avoid bubbles as centrifuging sub plates will result in a complete loss of transfection efficacy. This method can be applied to biological experiment, crimes, and transfection and is suitable for mostly that can be performed in a 384 well plate format. A fullproof transfection paves the way for new applications such as plasmid based known for crisper cast non-screening approaches enabling the monitoring of intermittent affect currently unable to be sorted in poor strategies.

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