Name: using the gene pulser mxcell electroporation system to transfect primary cells with high efficiency
Uploaded: 2010-01-07T00:00:00
Duration: 12 min 55 s
Description: Watch this Scientific Journal Video about Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency Video at JoVE.com

1) Cell preparation
 <ol>
 <li>When using adherent cells, it is necessary to trypsinize and collect the cells prior to electroporation.</li>
 <li>To compare transfection among the four mouse embryonic fibroblast (MEF) cultures, representing three different passage numbers, perform the following on each flask.</li>
 <li>Aspirate off cell culture media.</li>
 <li>Add PBS to wash cells.</li>
 <li>Remove PBS; add sufficient trypsin to cover cells, and wait a few minutes to allow trypsin to detach cells.</li>
 <li>Check flasks with a microscope to verify the condition of the cells, smack flask to detach cells, and then check flask again to make sure cells are all detached.</li>
 <li>Wait additional time and repeat if necessary.</li>
 <li>Once all cells are detached, add serum containing media to neutralize trypsin.</li>
 <li>Transfer cells to a centrifuge tube, and pellet cells by centrifugation (rcf = 300 x g).</li>
 <li>Remove supernatant and resuspend cells in a known volume of PBS. </li>
 <li>Count cells.</li>
 <li>Transfer to a new tube the appropriate volume of cell suspension to provide the required number of cells for experiments (you will need 150 &mu;L of cells per well at a density of 1 x 106 cells/mL).</li>
 <li>Centrifuge cells.</li>
 <li>Remove supernatant and resuspend cells in the appropriate volume of Gene Pulser electroporation buffer to achieve a cell density of 1 x 106 cells/mL.</li>
 <li>Add 20 &mu;g of plasmid per mL of cell suspension and mix gently.</li>
 </ol>
2) Electroporation vessel setup and electroporation
Plate setup and electroporation
<ol>
<li>Plug plate chamber into the power module of the Gene Pulser MXcell electroporation system. </li>
 <li>Pipette 150 &mu;L cell mixture or buffer into the wells of a 96-well electroporation plate.</li>
 <li>Put plate in plate chamber and pulse.</li>
 <li>Remove plate from chamber.</li>
 <li>Mix well contents by pipetting up and down in each well.</li>
 <li>Transfer the cells from each well to pre-warmed buffer in 12-well plates.</li>
 <li>Tap plate to distribute cells and put it in incubator.</li>
 <li>Let cells recover for 24 hours.</li>
</ol>
Cuvette setup and electroporation
<ol start="9">
 <li>Unplug plate chamber from the power module of the Gene Pulser MXcell electroporation system and plug in the ShockPod&trade; cuvette chamber.</li>
 <li>Pipette 600 &mu;L of cell suspension into a 0.4 cm gap electroporation cuvette.</li>
 <li>Put cuvette in ShockPod chamber and deliver electric pulse.</li>
 <li>Remove cuvette from chamber.</li>
 <li>Mix cuvette contents by pipetting up and down in cuvette.</li>
 <li>Transfer the cells from each well to pre-warmed buffer in 12-well plates.</li>
 <li>Tap plate to distribute cells and put it in incubator.</li>
 <li>Let the cells recover for 24 hours.</li>
 </ol>
3) Representative Results
After transfecting cells and allowing them to recover, analyze the transfection efficiency qualitatively, using epifluorescent microscopy, and quantitatively, using flow cytometry.
<img src="/files/ftp_upload/1662/1662fig1.jpg" alt="Figure 1" /> Figure 1. Cells that have been successfully electroporated and are now expressing the GFP gene appear under epifluorescent microscopy.
<img src="/files/ftp_upload/1662/1662fig2.jpg" alt="Figure 2" /> Figure 2. Viewing the cells under phase contrast allows visualization of both transfected and untransfected cells. These are the cells that were exposed to the lowest voltage electroporation pulse at 200V. The cells are largely confluent due to the high cell density.
<img src="/files/ftp_upload/1662/1662fig3.jpg" alt="Figure 3" /> Figure 3. The same field of view under epifluorescence shows a number of cells are expressing the GFP marker, but these are only a small percentage of the cells visible in the previous image.
<img src="/files/ftp_upload/1662/1662fig4.jpg" alt="Figure 4" /> Figure 4. At 250V, the total number of live cells seen under phase contrast decreases slightly.
<img src="/files/ftp_upload/1662/1662fig5.jpg" alt="Figure 5" /> Figure 5. Under epifluorescence, one can see that the number of GFP expressing cells has increased.
<img src="/files/ftp_upload/1662/1662fig6.jpg" alt="Figure 6" /> Figure 6. At the highest voltage applied, 375V, there are fewer live cells visible.
<img src="/files/ftp_upload/1662/1662fig7.jpg" alt="Figure 7" /> Figure 7. However, a large percentage of the remaining cells are expressing GFP. Which condition is optimal depends on the experimental design. In some experiments the largest number of transfected cells might be optimal, in other experiments the highest percentage transfection might be best.
We are interested in the percentage of cells that are GFP positive under each condition and how the percentages vary with cell age. Flow cytometry can provide quantitative information about the transfection results under each of the different electroporation conditions.
<img src="/files/ftp_upload/1662/1662fig8.jpg" alt="Figure 8" /> Figure 8. Here the percentage of cells that are GFP positive in the passage 5 cells under each of the 12 electroporation conditions are shown. The maximum transfection percentage was approximately 80% under the highest voltage exponential decay pulse, condition 6, and 70% under the strongest square wave pulse tested, condition 12.
<img src="/files/ftp_upload/1662/1662fig9.jpg" alt="Figure 9" /> Figure 9. With the cells passed 9 times prior to the electroporation, the overall pattern of transfection percentage is nearly identical, but with a very slight decrease in the transfection percentages.
<img src="/files/ftp_upload/1662/1662fig10.jpg" alt="Figure 10" /> Figure 10. Shown here are the percentages of GFP cells in the passage 13 cells which show a marked decrease in transfection percentage relative to the younger cells. The highest transfection percentages were approximately half what was achieved with the younger cells demonstrating the importance of using healthy cells as soon after isolation as possible.

 <table border="1">
 <tbody>
 <tr>
 <td colspan="3">Electroporation Conditions used for transfecting MEF cells using the Gene Pulser MXcell</td>
 </tr>
 <tr>
 <td>Condition (1-6) 
 Exponential Decay pulses, 
 all with 350 uF, 1000ohm </td>
 <td>Voltage (V)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1</td>
 <td>200</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2</td>
 <td>250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3</td>
 <td>300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>4</td>
 <td>326</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5</td>
 <td>350</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>6</td>
 <td>376</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Condition (7-12) 
 Square Wave pulses, 
 all with 2000 uF, 1000 ohm, and 1 pulse</td>
 <td>Voltage (V)</td>
 <td>Pulse Duration (ms)</td>
 </tr>
 <tr>
 <td>7</td>
 <td>200</td>
 <td>10 </td>
 </tr>
 <tr>
 <td>8</td>
 <td>250</td>
 <td>10</td>
 </tr>
 <tr>
 <td>9</td>
 <td>300</td>
 <td>10</td>
 </tr>
 <tr>
 <td>10</td>
 <td>200</td>
 <td>20</td>
 </tr>
 <tr>
 <td>11</td>
 <td>250</td>
 <td>20</td>
 </tr>
 <tr>
 <td>12</td>
 <td>300</td>
 <td>20</td>
 </tr>
 </tbody>
 </table>

The authors are employed by Bio-Rad Laboratories that produces reagents and instrument used in this article

This video article demonstrates how to use the MXcell electroporation system to easily identify optimal electroporation conditions for MEFs or other primary cell lines. The 96-well plate format allows for many replicates of experimental or optimization conditions to be performed simultaneously, which can eliminate the need for many separate experiments. While carrying out this procedure, one should to remember to use healthy cells as soon after isolation as possible and to use electroporation conditions that are matched to the electroporation buffer.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gene Pulser&reg; Electroporation Buffer</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2676</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser MXcell&trade; Electroporation System</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2670</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser MXcell&trade; ShockPod&trade; Cuvette Chamber</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2673</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser MXcell&trade; Electroporation System With ShockPod&trade; Cuvette Chamber</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2674</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

using the gene pulser mxcell electroporation system to transfect primary cells with high efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

Watch this Scientific Journal Video about Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency Video at JoVE.com

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency Video (Video) | JoVE

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Research

JoVE Journal

Biology

Electroporation of Mycobacteria

Electroporation of Mycobacteria Video (Video) | JoVE

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells Video (Video) | JoVE

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.  Video (Video) | JoVE

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells Video (Video) | JoVE

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye Video (Video) | JoVE

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

SiRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells Video (Video) | JoVE

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva Video (Video) | JoVE

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry Video (Video) | JoVE

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene Video (Video) | JoVE

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC Video (Video) | JoVE

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...