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

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.