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

Summary

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.

Abstract

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 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.

Introduction

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 "user-friendly" 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.

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. Please click here to view a larger version of this figure.

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 done^2,3,4. DNA plasmid cell transfection involves several steps to reach high efficiency, such as an initial complex uptake, endosomal escape, and cytoplasmic transport to the nucleus^5,6. 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 cytoxicity^8,9. 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 reagents^11,12,13, 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 µL 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' 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.

Protocol

1. Advance preparations

1. 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.
   1. For the 1 µL diluent dispense step, mount a 1 µL cassette and prepare a program with the settings as described in steps 1.1.1.1 and 1.1.1.2.
      1. Adjust the flow rate parameter to High for the best throughput as no biological material damage is expected in this step. Adjust the dispense height to 9.6 mm (according to the cell culture plate used, Supplemental Figure 1) to allow the 1 µL drop to touch the bottom of the wells during dispensation.
         NOTE: This step is crucial to avoid the retention of the droplets on the dispensing head until reaching a sufficient volume to drop.
      2. Adjust the plate clear height to 14.4 mm to allow a 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 and verify that the head is high enough to allow displacement of the head after dispensing each row.
         NOTE: Avoiding drop retention is a crucial parameter as it will impair the accuracy of the volume of the dispensation.
   2. For dispensing the 40 µL cell suspension, mount a 10 µL cassette and prepare a program with the settings as described in steps 1.1.2.1-1.1.2.2.
      1. Adjust the flow rate parameter to Low to dispense cells with a low speed to avoid promoting potential damages to the cells by shear stress and high impact on the bottom of the wells. Adjust the dispensation height to 11.43 mm (according to the cell culture plate used, Supplemental Figure 1), 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. Adjust the plate clear height to 16 mm to allow free displacement of the dispensing head over the plate after dispensing each row.
      2. Visually control the proper settings of the peristaltic liquid handler head height: make sure no drops are retained on the dispensing tips while dispensing and verify that the head is high enough to allow displacement of the head after dispensing each row.
         NOTE: Avoiding drop retention is a crucial parameter as it will lead to dispensing an unreliable cell number.
2. DNA plasmid preparation (classical miniprep extraction protocol)
   1. Grow a transformed DH5α bacteria strain in LB medium supplemented with 125 µg/mL ampicillin selection antibiotic (Table of Materials) overnight at 37 °C and under gentle agitation (200 rpm) on an orbital shaker (Table of Materials).
   2. Harvest 2 mL of the culture, pellet the cells by centrifuging for 5 min at 6,000 x g, and discard the supernatant.
   3. Resuspend the cell pellet with 250 µL of resuspension buffer containing RNase A (Table of Materials). Add 250 µL of lysis buffer and incubate for 5 min at room temperature, according to the manufacturer's instructions.
   4. Stop the lysis reaction by adding 300 µL of neutralization buffer (Table of Materials) and vortexting shortly. Centrifuge the tubes for 5 min at 11,000 x g.
   5. Place a new plasmid minicolumn (Table of Materials) in a 2 mL collection tube and decant the supernatant in the column by centrifuging for 1 min at 11,000 x g.
   6. Discard the flow-through and place the minicolumn back in the collection tube.
   7. Wash the plasmid minicolumn with 500 µL of optional washing buffer (Table of Materials) and centrifuge for 1 min at 11,000 x g, according to the manufacturer's instructions.
   8. Discard the flow-through and place the plasmid minicolumn back in the collection tube.
   9. Add 700 µL of washing buffer (Table of Materials) supplemented with ethanol and centrifuge for 1 min at 11,000 x g, according to the manufacturer's instructions.
   10. Discard the flow-through and centrifuge the plasmid minicolumn and its collection tube 1x more for 2 min at 11,000 x g to dry the silica membrane.
   11. Place the dried plasmid minicolumn in a new 1.5 mL tube and add 30 µL of distilled water prewarmed at 60 °C, incubate it for 2 min at room temperature, and then, centrifuge it for 1 min at 11,000 x g.
   12. Discard the plasmid minicolumn and keep the eluate containing the purified DNA plasmid.
   13. Measure the DNA concentration of the eluted DNA using a microvolume spectrophotometer (Table of Materials).
       1. Turn on the spectrophotometer and choose the DNA measurement settings.
       2. Raise the sampling arm of the spectrophotometer and pipette 1 µL of water onto the measurement pedestal to perform a blank calibration.
       3. Lower the sampling arm, start the blank measurement, and wait for completion.
       4. Raise the sampling arm and wipe the sample from the upper and lower pedestals.
       5. Pipette 1 µL of the DNA solution onto the lower pedestal to measure it.
       6. Lower the sampling arm, start the DNA concentration measurement, and wait for completion.
       7. Raise the sampling arm and wipe the sample from the upper and lower pedestals.
       8. For further DNA concentration measurements, repeat steps 1.2.13.5-1.2.13.7.
   14. Once the measurements are finished, store the DNA solutions at 4 °C until use.

2. Experimental design and generation of the picklists to drive the ADE-based dispenses

NOTE: A dedicated "user-friendly" spreadsheet macro was developed to manage DNA amounts and mix up to four plasmids in a 384-well plate format. Based on the entered experimental design, this macro generates the necessary files to drive the ADE-based DNA transfection protocol by nanodispenser. In order to generate these files, several fields have to be filled in the Template Sheet as shown in Figure 2.

Figure 2: Generation of the picklists to drive the ADE dispensation using the spreadsheet macro. Several parameters have to be filled, namely (1) the transfection reagent (TR) and the minimal/maximal volumes to be used in the source plate, (2) the initial plasmid concentrations to be dispensed in the source plate, and (3) the whole-plate design, including the expected plasmid amounts and multiplexing in each of the 384-wells. (4) Generate Picklists activation allows the different fields to be verified and, once properly filled, picklists for DNA and TR dispensation and the necessary source plate template are automatically generated. Please click here to view a larger version of this figure.

1. Enter the nanodispenser protocol parameters in the pink fields. Set the transfection reagent (TR) mixture value to 500 nL. Set the minimal volume value in the source plate wells to 4 µL. Set the maximal volume in the source plate wells to 11.25 µL.
   NOTE: The nanodispenser used here can only transfer a maximum of 500 nL in one run of ADE. These pink fields are prefilled with the recommended values but can be modified according to the user needs.
2. Enter 100 ng/µL DNA starting concentrations in the blue fields corresponding to the underlying DNA.
   NOTE: This value is the optimal concentration previously defined but can, however, be modified for different user needs.
3. Enter the desired DNA amount in the grey/green fields. Enter the amounts and plasmid names for the 384 wells, ensuring the same spelling if the same plasmid is used in several wells.
4. Generate the source plate design, the picklists files, and the pipetting guide file. Click on Generate Picklists to allow the macro to generate the DNA-Picklist.csv, the T.R.-Picklist.csv and the 384-Wells-Pipetting-Guide.csv files from data collected on the corresponding sheet. If requested, correct the orange-filled cell values as it indicates errors or volumes that cannot be handled by the nanodispenser.
5. Print the template(s) from the Source Plate sheet. Plasmid names and minimal volume to fill in the wells are indicated. Likewise, transfection reagent mixture volumes that will next have to be filled in the following wells are indicated as TR and highlighted in green.

3. DNA source plate preparation using the 384-well pipetting guide application

1. Dilute the stored DNA plasmid from step 1.2.14 to 100 ng/µL using distilled water.
2. Calibrate the 384-well grid to the plate dimensions: open the 384-well pipetting guide application on a tablet (Figure 3). Place the source plate on the grid on the lower screen, and in the upper left calibration menu, click + or - (or use the red cursor) 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.

Figure 3: Use of the 384-well pipetting guide application. (1) Calibration of the 384-well grid to the plate size; (2) ) Mount of a universal 3D-printed plate adapter to the tablet using double-sided tape; (3) Placement of the plate on the adapter; (4) Displacement of the grid to center it to the mounted plate. (5) Lock of the calibration step. (6) Opening of the 384 wells pipetting guide.csv file. (7) Given the file list, the application will indicate the expected source plate name, reagent (DNA or transfection reagent), the concentration, and the volume to dispense into the target wells, which will be illuminated one by one. (8) Left and right arrow buttons allow the user to follow the pipetting guide to easily dispense the reagents according to the spreadsheet macro source plate template(s). Please click here to view a larger version of this figure.

3. 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 on the screen to the plate position. Once the grid and well sizes are properly calibrated and located, tick the Lock calibration box.
4. Click on FILE and open the 384 wells pipetting guide.csv file. Follow the screen instructions to manually dispense the indicated volume of the indicated plasmid at the indicated concentration in the white highlighted well corresponding to the proper target destination of the expected plate. Use - or + arrows to go back or further in the DNA dispensing process. Stop dispensing when reaching the first Transfection reagent solution to load.
5. Once the DNA dispensations are finished, remove the source plate from the adapter. If several source plates have to be filled, then place a new source plate on the adapter and follow the dispensing instructions. Once the DNA dispensation has finished, centrifuge the DNA-filled source plate(s) (at 1,500 x g for 2 min) to ensure proper liquid leveling and to remove bubbles leading to inaccuracy in the ADE-based transfers.

4. Peristaltic liquid handler-based 1 µL diluent dispensation in the destination plate

NOTE: Perform steps 4.1-4.5 in a biological safety cabinet.

1. Disinfect the 1 µL cassette head by spraying it with a spray disinfectant (Table of Materials), and allow this solution to enter the tip holder. Absorb the remnant disinfectant on absorbing paper. Mount the 1 µL cassette on the peristaltic liquid handler device. Turn on the device and make sure the cassette type setting is correct (1 µL), as well as the plate format (384 wells).
2. Disinfect the entire lumen of the tubing: insert the tube organizer (holding the eight tubes together) in a sterile vessel and fill it with 5 mL of 70% alcohol. Using the priming function of the peristaltic liquid handler, first flush the alcohol in the tubing and then rinse it by passing 5 mL of distilled water and 5 mL of serum-free medium (Dulbecco's modified Eagle's medium [DMEM] supplemented with 100 U/mL penicillin-streptomycin; Table of Material), successively filling in the same vessel. Make sure none of the tip is clogged by visually inspecting the liquid flow from all of them.
3. Prime the tubing with serum-free medium by filling a new sterile vessel with 10 mL of prewarmed serum-free medium and diving the tube organizer in it. Press the prime button of the peristaltic liquid handler for about 10 s. Once again, ensure no tip is clogged by visually inspecting the liquid flow from all of them.
4. Fill the plate with 1 µL of diluent. Place a sterile 384-well culture plate (destination) on the peristaltic liquid handler plate carrier and remove its lid.
5. Run the precalibrated program to dispense 1 µL in each well of the 384-well plate. The dispensing time is approximately 8 s. Then replace the lid of the 384-well plate.
   NOTE: Alternatively, this step can be handled manually, in a safety cabinet, using a multichannel micropipette.

5. Performance of a survey to control the manually dispensed volumes

NOTE: For details, see Figure 4.

1. Run the nanodispenser program, go to the diagnostic tab, tick the source plate Out box, 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.
2. 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 µL as this will avoid transfers.

Figure 4: Defining the survey software parameters. (1) Start the nanodispenser program. (2) Open the Diagnostics tab. (3) Insert the source plate by ticking Out for the source plate and, then, In. (4) Define the source plate type in the menu when prompted. (5) In the Miscellaneous box, select Survey in the drop-down menu. (6) Launch the survey program by clicking on Launch. (7) Select the prefilled wells to measure. (8) Start the analysis by clicking on Go. (9) Once the survey is performed, the measured volumes are written in the corresponding selected wells. Please click here to view a larger version of this figure.

6. ADE-driven DNA dispensation into the destination plate

1. Run the picklist software, set the 384-well source and destination plate types to 384_LDV and Greiner 384PS_781096, respectively (Figure 5). Set the device to aqueous buffer dispensing mode by selecting 384LDV_AQ_B2, and untick "optimize transfer throughput”.

Figure 5: Performance of the picklist-based dispensations. (1) Start the nanodispenser software. In the Protocol tab, select (2) the sample plate format, (3) the destination plate type and (4) unticks "optimize transfer throughput". (5) Select the Pick List tab. (6) Click on Import and select the proper *.csv file (DNA-PickList or T.R.-Picklist). (7) Once selected, click on Import. (8) Click on Play and save the protocol. (9) Perform a dispensation simulation by clicking on Simulate, or (10) Start the programmed dispensation by clicking on Run. Please click here to view a larger version of this figure.

2. Select "Pick List" tab, click on Import, select the DNA-Picklist.csv file. Click on Play and save the protocol. Click on Simulate to perform a simulation of the programmed dispensations to make sure that the picklist matches the expected experimental design. Once completed, click on Close.
3. Click on Play, and then Run, to start the dispensing program: when asked, insert the requested source plate (DNA solutions manually filled) and the destination plate (diluent-filled) in the nanodispenser. 
   NOTE: The dispensing time is approximately 5-20 min for a complete 384-well plate, depending on the selected volumes and the total number of dispensations in the experimental design.
4. Alternatively, pause the protocol here as the diluent- and DNA-filled plates can handle dry or frozen storage for up to 7 days. For dry storage, let the plates dry on the bench at room temperature and then store them the same way. Thaw and centrifuge (at 1,500 x g for 2 min) frozen stored plates before use in a transfection step (section 7).
    

7. ADE-driven transfection reagent dispensation

1. In a biosafety cabinet, extemporaneously dilute lipopolyplex transfection reagent in serum-free medium to a 1x final concentration. Vortex and immediately dispense this transfection reagent mix according to the predefined source plate(s) designed by the macro and using the precalibrated 384-well pipetting guide application as described in step 3.4.
   NOTE: Do not centrifuge the source plate once it is loaded with the reagent as no transfection is noticed after centrifugation.
2. Run the nanodispenser program to perform a "survey" as described in section 5, in order to control the volumes of all the manually filled TR wells of the source plate(s) to avoid dispensing errors due to volumes exceeding 12 µL.
3. Click on Reset to clear the sample list of the DNA picklist in the picklist software, and verify that the device parameters are still set to aqueous buffers and to the source and destination plate types used, as in step 6.1.
4. Click on Import and choose the TR-Picklist.csv file. Click on Play and save the protocol if prompted, and (this is optionally but strongly recommended) perform a simulation of the programmed transfection reagent mixture dispensations to ensure proper design of the dispensations by clicking on the Simulate button. Once completed, click on Close.
5. Click on Play, and then Run button to start the dispensing program: as requested, place the source plate(s) (TR-mixture-filled) and the destination plate (diluent- and DNA-filled) in the nanodispenser.
   NOTE: The dispensing time is  less than 20 min for a complete 384-well plate when dispensing 500 nL of TR mixture.
6. Incubate 15-30 min at room temperature after adding the TR to the DNA as indicated by the manufacturer's protocol.

8. Peristaltic liquid handler-based cell dispensation

1. Prepare the peristaltic liquid handler for dispensing cells. Disinfect a 10 µL cassette head by spraying it with Aniospray Surf 29 Disinfectant and absorbing the remnant on paper. Mount the cassette on the peristaltic liquid handler device, change the cassette type setting to 10 µL, and make sure the plate format is set to 384 wells.
2. Disinfect the 10 µL cassette tubing as previously described in step 4.2. Dive the tube organizer in a sterile vessel and flush the tubing with 5 mL of 70% alcohol, then with 5 mL of distilled water, and finally, with 5 mL of serum-free medium, successively filled in the same vessel and until each tube is empty.
3. Prepare the cell suspension to dispense. From a confluent HeLa cell B10-culture dish, wash the cells 1x with 1x phosphate-buffered saline (PBS) solution, and then dissociate the cells with trypsin/EDTA for 5 min at 37 °C.
4. Verify the cell dissociation under a microscope and stop the trypsin/EDTA action by adding 10 mL of complete medium (DMEM supplemented with 10% fetal bovine serum and 100 U/mL penicillin-streptomycin; see the Table of Material) in the culture dish. Harvest cells in a 50 mL tube and count the cells under the microscope, using a Malassez cell or an automatic cell counter.
5. Prepare at least 25 mL of HeLa cell suspension at a concentration of 37,500 cells/mL in complete medium (i.e., 1,500 cells/40 µL) for a complete 384-well plate, to ensure tube priming and 40 µL/well dispensation.
6. To dispense the cells, fill a new sterile vessel with the prepared cell suspension and stir it to avoid sedimentation leading to inaccuracy in the cell density of the dispensation. Insert the tube organizer in this solution and press the Prime button until the cell suspension is starting to flush from the dispensing head. Make sure none of the tip is clogged by visually inspecting the liquid flow from all of them, and ensure each tube is loaded with cell suspension.
7. Load the DNA and TR-filled 384-well destination plate on the peristaltic liquid handler plate carrier and remove its lid. Run the precalibrated program to dispense 40 µL of the cell suspension on the complete 384-well plate (i.e., 1,500 cells/well). The dispensing time is about 8 s. Replace the lid of the 384-well plate.
   NOTE: Alternatively, the 40 µL cell suspension can be manually dispensed using a multichannel micropipette.

9. Custom biological assay (cell transfection efficiency monitoring)

NOTE: Following the experimental settings and intent of the experiment, use the required methods for luminescence, fluorescence, high-content screening, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). In this section of the protocol, cell transfection efficiency is evaluated by automated fluorescence microscopy and image analysis.

1. Incubate the plate at 37 °C with 5% CO₂ in a water-saturated atmosphere and until proper protein expression.
   NOTE: Here, a 48 h incubation time is used for HeLa cells to monitor the transfection efficiency, using tdTomato- and mVenus-expressing plasmids.
2. Remove the culture medium 48 h post-transfection by inverting the plate, add 30 µL/well of 10% formalin using the peristaltic liquid handler (10 µL cassette), and incubate for 15 min at room temperature.
3. Remove the formalin by inverting the plate; then, incubate the cells for 15 min at room temperature with 0.1 ng/mL Hoechst diluted in 1x PBS solution.
4. Wash the cells 3x for 15 min with 80 µL of 1x PBS adjusted to pH = 8 in order to recover the high fluorescence signal lost by the 6.9 pH of the formalin solution incubation step.
5. Using an automated fluorescent microscope, acquire images of two or three fluorescent channels (Hoechst, tdTomato, and mVenus) sequentially with 10x objectives and a proper emission filter set (4′,6-diamidino-2-phenylindole [DAPI], dsRed, and fluorescein isothiocyanate [FITC], respectively).
6. To evaluate transfection efficiencies, use image analysis software to determine the transfection efficiencies using script analysis based on nuclei staining.

Representative Results

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 transfection observed in preliminary experiments (i.e., only in the center of the wells). As shown in Figure 6A, the transfection of HeLa cells using lipopolyplex reagent²⁰ was successful. Interestingly, by using a 1 µL diluent volume, DNA amounts ranging from 5 to 30 ng showed the same efficiency and up to 90% cell transfection compared to higher amounts, such as 50 and 100 ng, for which an abrupt decrease was observed. We tried various diluent volumes ranging from 15 nL to 4 µL and identified 1 µL to be the best condition, as significantly exemplified here using 30 ng of DNA.

Figure 6: Representative results. (A) Impact of the DNA amount and diluent volume on the transfection efficiency. HeLa cells were reverse transfected using the nanodispenser device and lipopolyplex, using a 1x concentration as recommended by the manufacturer. Of the recommended diluent (serum-free medium), 15-4,000 nL was used with 10-100 ng amounts of red-fluorescent-expressing plasmid (tdTomato). Transfection efficiencies were determined 48 h post-transfection using image-based analysis software. The results are expressed as a percentage of transfected cells for the increasing DNA amount, and the diluent volume shows the optimal conditions: 30 ng of total DNA with an increasing diluent volume and 1 µL of diluent with increasing DNA amounts. The error bars represent the SEM with n ≥ 4. Two-way ANOVA and Bonferroni post-test were used for statistical analysis. *p < 0.05 compared to other dots. (B) Stability of the prepared DNA plates. Diluent (1 µL) was dispensed using the peristaltic liquid handler, and 30 ng of DNA was dispensed and immediately transfected using lipopolyplex reagent dispensed by ADE (control) or either stored at room temperature once dry or frozen at -20 °C. At days 0, 2, or 7, dry DNA was rehydrated with 1 µL of diluent dispensed using the peristaltic liquid handler, and frozen plates were thawed at room temperature and centrifuged (at 1,500 x g for 2 min). Cells were then seeded using the peristaltic liquid handler according to the described protocol. The error bars represent the SEM with n ≥ 3. Two-way ANOVA and Bonferroni post-test were used for statistical analysis. ns = nonsignificantly different. (C) Plasmid DNA cotransfection efficiency. HeLa cells were transfected with 30 ng of mVenus- and tdTomato-expressing plasmid loaded in two separate source wells (using a 1.7 ratio of mVenus over tdTomato in order to level their relative fluorescence output). Transfection efficiencies were compared 48 h post-transfection using image-based analysis software and were expressed as a percentage of transfected cells and a percentage of cotransfected cells within the transfected population. The percentage of cotransfected cells was determined by calculating the green-fluorescence-expressing cell number in the red fluorescent population cells. The error bars represent the SEM with n ≥ 3. Two-way ANOVA and Bonferroni post-test were used for statistical analysis. ns = nonsignificantly different. (D) Representative fields of fluorescence microscopy from the image acquisition shown in panel C using three fluorescent channels (Hoechst, tdTomato, and mVenus) sequentially acquired by an imaging platform (Table of Materials), using 10x objectives and a proper emission filter set (DAPI, dsRed, and FITC, respectively). This figure has been modified from Colin et al.¹. Please click here to view a larger version of this figure.

In order to further enhance the throughput of this protocol, we next examined if a source plate storage prefilled with DNA and diluent solutions could be stored and used at a later stage. Two ways of efficiently storing DNA were tested, namely dry storing the plate by letting it dry on the bench or frozen storage (at -20 °C). Both storage methods did not lead to significantly different results than freshly dispensed DNA solution stored for up to 7 days (Figure 6B), and both methods made it possible to perform transfection from stored DNA prefilled plates, such as a bank of plasmids.

Finally, as plasmid transfection most often occurs using at least two different plasmids, we next examined the DNA multiplexing ability of the protocol presented here using the best identified conditions (1 µL of diluent and 30 ng of DNA). 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 (Figure 6C) showed the transfection efficiency to be about 80%; however, in the red population, nearly 100% of the cells were also cotransfected with the mVenus-expressing plasmid as can be seen in the representative software-based image analysis of Figure 6D.

Supplemental Figure 1: Diagram showing a suitable dispense height for the drop to touch the bottom of the well to avoid its retention on the dispensing tip. On the left, proper settings allow the drop to spread on the well surface avoiding its retention on the dispensing tips. On the right, bad settings lead to droplet retention that can be observed during the head movement to the next raw. Please click here to download this figure.

Discussion

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 varying the cell number, diluent volume, total DNA amount, and transfection reagent nature, concentration, or even volume used as was the case during the optimization of this protocol for HeLa cells¹.

The overall protocol presented here has been developed and further optimized to allow cell transfection even by novices in the field. In order to reach this goal, key tools to render the protocol as simple as possible and avoid human errors have been developed: a user-friendly spreadsheet macro to easily design the experiment and a tablet application to guide the user to properly fill the source plate(s).

Thus, to ensure the reliability of the protocol, only a few critical steps have to be controlled: i) a proper experimental design; ii) the proper peristaltic dispensations from the classical peristaltic liquid handler; iii) the proper DNA and transfection reagent acoustic-based dispensations; iv) avoiding the centrifugation of the source plate before the transfection reagent dispensation as that seemed to impair transfection. Following these few recommendations would ensure the efficient transfection of the cells.

Proper experimental design

The experimental setup has been rendered user-friendly by the development of the macro spreadsheet which just has to be filled with the expected DNA plasmids name, desired amount,  and some key parameter values. Once filled, the macro first analyzes the entered parameters to detect potential errors, such as suitable minimal and maximal volumes entered for the source wells and the transfection reagent dispensation volume. Furthermore, based on the DNA concentrations entered in each of the four possible rows and the plasmid quantity entered in the underlying fields, the macro verifies if the expected volumes to dispense are multiples of 2.5 nL (volume of the drops dispensed by the nanodispenser). Once a check for any errors has been performed, the macro calculates the total amount of each DNA sample that will have to be dispensed and, then, designs the source plate template (by sorting the DNA names in alphabetical order). The volumes indicated in the source plate(s) take into account the working volumes expected in a source well (calculated from the minimal and maximal volume values filled in the template sheet). All the expected DNA dispensations in each of the wells are then written on the DNA-picklist sheet. The list of DNA-transfected wells is then used to write the TR-picklist sheet using the transfection reagent volume indicated on the template sheet. The volumes calculated on the source plate sheet are then transferred to the 384-well pipetting guide sheet. Data from the DNA-picklist, TR-picklist, and 384-well pipetting guide are then used to generate the corresponding files in the *.csv format.

Proper DNA and TR dispensation on the source plate

As dispensing on a 384-well source plate and, more specifically, locating the target well can promote errors and are furthermore time-consuming, we have developed a dedicated tablet-based application similar to iPipet²¹. Unlike iPipet, the one described here can be used with Android (only Android version 4.4 and up is supported). Based on the 384-Wells-Pipetting-Guide.csv file generated by the macro spreadsheet, it helps the user in the overall dispensing process. Whereas its use for a few dispensations is not worth it, it can be interesting to save time and avoid errors if a large number of DNA and TR dispensations are expected. The .csv file must contain well, plate, name, concentration, and volume information. This application could then be used for other applications, such as dispensing solutions (reagents, dye, compound, etc.) in the target well, according to a user dedicated csv file. Furthermore, it allows the user to illuminate an entire row or line by entering the relevant information into the target well column using this expected format: Row_1 (to 24) or Line_A (to P).

Troubleshooting poor cell transfection efficiency

Several parameters described below may impair cell transfection in the described ADE-based protocol and would have to be individually checked and circumvented in case of efficiency problems.

One of the first important parameters for transfection is the quality of the cells and the density used during sowing. Although each cell type will require different parameters, some of them must be respected to ensure a successful transfection. First of all, the cell suspension must be prepared extemporaneously from a subconfluent plate to avoid cell stress before transfection, and they should not be left lying on the bench for too long (2 h maximum). Second, the seeding density of the cells must be low enough for two reasons: to avoid cellular contacts and promote a high cell surface once spread but also because actively dividing cells better take up foreign nucleic acid^22,23. Unfortunately, the optimal cell density for transfection varies depending on cell types and transfection technology and has to be determined for each cell line. These are crucial parameters for ensuring effective transfection.

Another important parameter that can modulate transfection efficiency is the cell passage number²⁴. Indeed, cells in culture are continually subjected to evolution due to competition and natural selection. It is well known that differential gene expression between low and high cell passage numbers is expected in most cell lines due to dedifferentiation as the passage number increases. Following this phenomenon, transfection efficiency may also be affected. However, passage-related effects have been demonstrated to be heavily dependent on the cell line and the culture conditions. On top of that, what is considered a "high" passage level varies from one cell line to another. This means that the passage number range under which a set of experiments can be reliably performed has to be determined for each given cell line.

Another parameter which enhances the difficulty in determining the best conditions for transfection is that culture medium composition also plays a crucial role since the presence of serum and/or antibiotics modulate transfection efficiency. Indeed, most commercial protocols recommend the use of serum-free medium during the transfection step to enhance efficiency or circumvent problems of poor efficiency^3,25,26,27. However, this parameter is indeed more complex to apprehend as it has been shown that, for a given cell line, early versus late passages may enhance or lower efficiency depending on serum presence or absence in the culture medium²⁸. Other researchers preconize the use of antibiotic-free medium for the passage before the transfection when culturing cells, in order to obtain high-quality cells for transfection²⁹. To conclude, when optimizing transfection conditions for a given cell type, each of these parameters should be tested: early or late passage cells and the use of medium with or without serum during the last passage before harvesting the cells and/or during the transfection step itself.

During the optimization of the protocol presented here, two kinds of reagent were used: a liposomal reagent forming liposome-like complexes and lipopolyplex reagent, a nonliposomal polymeric compound¹. Whereas we had success for years manually transfecting HeLa cells with the first one, poor transfection efficiency was observed in the current automated protocol. This was probably due to a required vortexing step when mixing DNA with transfection reagent that cannot be performed in the 384-well plate format. The lipopolyplex does not need such a step and, therefore, led to higher transfection efficiencies in all the settings tested. Although this has not been confirmed in the current study, avoiding transfection reagents that require a physical mixing step such has pipetting or vortexing will probably lead to better results.

We also recommend the use of a transfection reagent compatible with reverse transfection as the presented protocol is based on a reverse transfection. Some cells are known to be difficult to transfect and some dedicated chemical compounds are developed to promote higher transfection efficiency^30,31. If aiming at transfecting hard-to-transfect cells, we recommend testing the given cell-type or cell-line-dedicated transfection reagents, assuming reverse transfection is feasible with these ones.

Several parameters detailed in the underlying paragraphs may have an impact and somewhat impair the ADE dispensing process, possibly ruining the final experiment.

The nanodispenser was developed to dispense 2.5 nL droplets from source wells filled with 3-12 µL of aqueous buffer (i.e., 9 µL working volume). The nanodispenser device used in this study integrates a dynamic fluid analysis technology for the determination of the fluid composition and liquid height in the source plate in order to control the power needed to eject 2.5 nL droplets¹⁹. While filling the source plate manually or using a classical peristaltic liquid handler, the volumes are most often not so accurate than those determined by the dynamic fluid analysis. This is a crucial point to take into account as the device is not able to dispense from source wells loaded with more than 12 µL. Thus, their presence would compromise the experiment. Of course, a list of the performed dispensations can be generated at the end of the program but this requires the user to adjust volumes and run a program to recover the missed dispensations only. To avoid these disagreements, it is recommended to perform a "survey" once the DNA plasmids have been loaded to verify the expected volume in each concerned well.

A crucial parameter for droplet ejection is the variation in liquid surface tension^17,32. Water DNA solutions are known for their viscosity; this might impair the dispensing process by ADE. Whereas the physical parameters were not determined in our previous study¹, higher DNA solution concentrations in the source plate resulted in lower transfection efficiency, even for the same total amount of DNA dispensed, probably because of this phenomenon. Thus, we recommend using a plasmid dilution of 100 ng/µL for best results, although other concentrations might be tested for user convenience. To ensure proper ADE with the user-needed DNA concentration, a dye can be added to the solution to monitor the droplet ejection in the wells or, even better, on the plate lid. As the presented protocol's first goal was to gain high throughput, cheap minicolumn-based plasmid DNA minipreparation kits compatible with plate-based high-throughput plasmid purification protocols were used. Whereas it worked properly during the performance of the experiment, in case of low efficiency and poor cell viability after transfection, it is recommended to use higher-grade DNA purification methods such as midi- or maxi-preparations or even endotoxin-free commercially available kits that would ensure a better DNA purity and lower toxicity for the cells³³.

Troubleshooting inhomogeneous cell transfection over the well surface

First attempts when setting up the protocol led to cell transfection only in the center of the wells, where the drops were sent by ADE. Indeed, we noticed that the DNA and transfection mixture was not spreading all over the well surface and was drying before cell addition due to the low volumes dispensed (barely 500 nL). To circumvent this problem, a diluent dispensing step was added to allow the DNA/TR mixture to spread all over the wells before cell addition. This resulted in a homogeneous cell transfection in the well. Thus, when reproducing the experiment and the DNA/TR mixture does not spread all over the wells, the diluent volume may be adjusted according to the user's needs.

As the acoustic liquid handler can distribute volumes in the nanoliter range, the described method potentially does not have any technical limitations. However, we noticed that aqueous-solution-filled wells are subject to evaporation, which could represent a limitation. If the exact volumes to be dispensed are lowered by this evaporation, the nanodispensing will not be performed to the expected end. In this case, an error report is generated by the nanodispenser. To circumvent this problem, use higher volumes than expected while staying in the acceptable upper range (indeed, lower than 12 µL). However, if evaporation leads to the impairment of some dispensations, an error report is generated by the nanodispenser. This file can be used to fill the source plate with new reagent to be dispensed on the concerned destination wells. Doing this in a short time interval did not seem to impair the transfection efficiency.

The protocol described here is the first one to obtain such throughput for independent DNA plasmid transfections. Best rates reached before were for 288 different conditions that required highly specialized skills to be performed simultaneously¹⁵. This, apart from its high throughput, the current protocol has other significant advantages as the overall process has been optimized to allow its use by nonspecialists and tools have been developed to avoid errors, namely i) a dedicated spreadsheet macro allowing the easy design of the experimental template, ii) the automatic generation of the corresponding DNA and transfection reagent source plate(s) template by this macro, iii) the generation of two ready-to-use files to control the software-driven dispensations of DNA and transfection reagent by the nanodispenser device, and iv) the export of a "384-well pipetting guide" file corresponding to the source plate(s) designed, to be used by a dedicated tablet-based application also developed in order to avoid human errors while dispensing in the 384-well source plate(s).

Future improvements of the protocol

In order to enhance throughput and reproducibility, the source plate filled with plasmids could be stored at 4 °C or frozen as usual for DNA stock solutions. Furthermore, we showed that the preloaded destination plate prefilled with DNA can also be stored dry or frozen for at least 7 days before adding the transfection reagent and cells, leading to the enhancement of the overall throughput and ease of the protocol. DNA conservation for more than 4 years has previously been reported using optimized media³⁴ and should, therefore, be tested in the context of this protocol as it will push it a step further, enabling the long-term storage of plates prefilled with banks of plasmids and ready-to-use in transfection experiments.

We previously showed that the identified optimal transfection conditions could be transferred to higher-scale experiments, from 96-well to 10 cm culture dishes¹, by calculating the DNA amount, transfection reagent volume, and cell density that should be used based on the optimized 384-well plate protocol. As 1536-well plate dispensation is manageable by the nanodispenser too, the protocol can also be performed at a lower scale, thus enhancing its throughput. However, a major limitation to reach this format is the ability to dispense cells and manage the final  read-out in such a miniaturized format. Cell dispensing by ADE has already been successfully performed in the 1536-well plate format³⁵ using a solution of neutral density that prevented cell seeding and ensured equal cell density in time. Based on the cell number used here and the surface ratio of 384 (0.056 cm²) versus 1536 wells (0.025 cm²), 500-650 cells would be dispensed in this last format. Such a cell dispensation number range has been shown to be highly reliable if 14%-18% concentrated solution of neutral density is used. Under these settings, 100 nL of cell suspension would be distributed over 1536 wells. With a working solution of 5-8 µL in such wells, adding 5-8 µL of culture medium using classical peristaltic liquid handlers would dilute the antiseeding solution to less than 0.3%, thus allowing proper cell seeding. Transfecting cells on such a low scale thus seems technically possible; however, effect of the residual concentration of the neutral density solution on transfection efficiency remains to be determined by further work.

Using source plate types bearing higher working volumes to dispense cells, such as 384-well polypropylene source plates having a working volume of 45 µL, would allow 100 nL cell solution dispensations from only four source wells for an overall 1536-well plate. Furthermore, new nanodispensers are able to dispense drops of 25 nL instead of the 2.5 nL that was used in this protocol. This drop size, then, divides 10-fold the dispensing time but imply to use multiple of 25 nL volumes which, however, stay compatible with the different volumes dispensed in the protocol presented here.

Based on these latest works and technological improvements, a further ADE cell dispensing step to achieve a 1536-well plate transfection could be easily added to the current protocol. However, reaching such miniaturizations is only worth it if the bioassay is concomitantly feasible in such a miniaturized format.

In conclusion, we developed an easy, high-throughput and accurate transfection method bearing several advantages due to miniaturization: (1) lowering the costs of transfection reagent; (2) reducing the waste of DNA preparations; (3) ensuring that even beginners can successfully perform cell transfection. Indeed, it requires only a few easy manual steps, namely diluting DNA to 100 ng/µL, dispensing it on a source plate (one plasmid/well) according to the spreadsheet-generated template and using the tablet-based pipetting guide, and preparing the cell suspension before seeding. The ADE-based nanodispenser is in charge of the time-consuming and error-prone dose delivery and multiplexing of the plasmids, according to the given template of the experiment.

Furthermore, whereas this protocol could ensure most of the basic biological intents of classical transfection experiments, it could also open up new ways for array-based experiments. For example, expressing or knocking down each human protein-coding gene from the human ORFeome collection³⁶ or CRISPR-Cas9 library-based approaches³⁷, respectively, would require less than 24 h on a dedicated automated platform (53 x 384-well plates), rather than 2-3 days of human work, assuming the use of a DNA-preloaded plate bank. Due to its high efficiency and high-throughput performance, the protocol presented here might even be able to achieve new nonpooled approaches for CRISPR-Cas9 based-studies with gRNA libraries/CRISPR-Cas9-expressing plasmids. Indeed, mild cellular phenotype changes, which currently cannot allow the required cell-sorting step, would finally be manageable, as one well would represent one knocked gene.

Materials

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