Neon® Transfection System

The following is a general protocol for using the Neon® Transfection System, along with specifics for optimized use with common cell lines found in the Heit Lab.

Introduction to Transfection with the Neon

The Thermofisher Neon® System is a high-efficiency, simple and versatile next-gen electroporation device. In short, the Neon® uses high-voltage pulses to interfere with the natural electrical potential of mammalian cells, allowing for entry of extracellular nucleic acids and proteins. The system includes the Neon® device, a pipettor, proprietary electrolytic buffers and special gold-plated pipette tips. While electroporation with the Neon® is a simple procedure, it is important to take great care when using the system to avoid damage or wastage.

General Neon® Protocol

Note: Before beginning the transfection procedure, it is a good idea to perform all the necessary calculations for maximum work efficiency, to minimize
the amount of time that your cells have to spend at room temperature.

1. Count cells using haemocytometer (and optionally Trypan blue)
2. Aliquot a volume of cell suspension that contains the appropriate number of cells for your cell type (see Table below for more info) into 1.5 ml microcentrifuge tube
3. Pellet the cells at 300-500xg for 3-5 minutes at room temperature.
   • During the spin, set the voltage parameters on the Neon® device. Place an electrolytic chamber (plastic cylinder) into the pipette station, firmly, until you hear or feel a click. Add 3 mL of electrolytic buffer E2 to the chamber, and be careful not to spill into the base of the station.

     

4. Remove and discard supernatant
• For established adherent/suspension cell lines: resuspend in 15-20 µL of buffer "R" + 1-5 µg of DNA/RNA/protein
• For primary blood-derived suspension cell lines: resuspend in 15-20 µL of buffer "T" + 1-5 µg of DNA/RNA/protein

Note:
• DNA concentration should be as high as possible, but DO NOT use ethanol precipitation to increase concentration of DNA that is to be used for electroporation.
• For low concentration solutions of DNA, do not exceed a 1:1 ratio with the appropriate electrolytic buffer
• Buffers "R" and "T" cannot be interchanged as differences in their chemical properties affect the amperage of electroporation and will reduce cell viability 
  if mis-used

5. Place a pipette tip onto the Neon® pipettor by depressing the plunger to the final stop, using the pipettor "claws" to grip the gold electrode, and pressing the pipettor firmly onto the tip such that the plastic skirting of the tip is flush with the bottom part of the pipettor handle (see figure below)

   

6. Pipette the cell+DNA/RNA/protein suspension into the pipette tip.

AT THIS POINT, IT IS IMPERATIVE TO LOOK CLOSELY AT THE TIP AND INSPECT FOR AIR BUBBLES (BOTH ABOBE AND BELOW THE PISTON). ANY AIR BUBBLES, NO MATTER THE SIZE, WILL CAUSE ARCING IN THE TIP, KILLING CELLS AND REDUCING THE TIP'S INTEGRITY & RE-USABILITY. In order to remove stubborn air bubbles, try rinsing the tip with ddH₂O

7. Once the tip is confirmed bubble-free, place the pipette into the chamber (containing electrolytic buffer E2) and press down on the pipettor (DO NOT depress the plunger) until it clicks into the chamber.
8. The moment of truth! Press the green "START" button on the Neon® device screen. You should hear some beeping and clicking, followed by a "Complete!" message.
   • Troubleshooting: If the machine displays an error message about checking for air bubbles, perform the following three checks. Ensure the electrolytic chamber is properly aligned and clicked in. Ensure the pipettor is clicked into the chamber all the way. Check that the tip is completely filled with cell suspension and contains no air pockets or bubbles: dispense the cell suspension back into the microcentrifuge tube and carefully re-uptake with the pipette, being extra vigilant to avoid air bubbles – the tip is made of flexible polypropylene and can be gently bent against the bottom of the tube if this is necessary. Sometimes the surface tension of the cell suspension will prevent it from entering the tip at all. If you suspect this is happening, uptake the cell suspension by slowly and gently feathering the plunger up and down while drawing up the suspension.

Cell-Specific Protocols

Cell Numbers and Voltage Parameters	Number of Cells to be taken from Culture Flask	Resuspension Buffer ("R" or "T")	Optimized Voltage/Pulse/Times
RAW 264.7	0.9-1.2x106 (conc: 6x107 cells/mL)	"R"	1680V/30ms/1
THP-1	3-4x105 (conc: 2x107 cells/mL)	"R"	1600V/10ms/3
J774.2	2.5-3x104 Note: small/invisible pellet (conc: 1.5x106 cells/mL)	"R"	1900V/30ms/1
Primary Murine Macrophage (C57)	5-8x105 (conc: 4x107 cells/mL)	"R"	1000V/40ms/2

Cleaning and Storage of Tips

The Neon® pipette tips can be re-used multiple times if cleaned and stored properly. It is not recommended to re-use tips across cell or DNA types, especially when doing important lab work.

When working with siRNA or CRISPR, always use a new tip!

To degrade DNA in the tip:

1. Aliquot 9 µL of DNase I Reaction Buffer into a microcentrifuge tube (found in TC fridge, on tube rack)
2. Add 1 µL of DNase I enzyme to reaction buffer (found in TC freezer). Do not let the enzyme stock temperature rise too much above -20^oC!
3. Pipette the DNase reaction mixture into the Neon® tip and allow it to incubate at room temperature for 15-20 mins
4. Rinse the tip once with DNase I Stop Solution

Tip storage

1. Rinse the tip 3 times with ddH₂O, then 3 times with 70% ethanol. Air dry the tip by pumping the plunger 10-15 times
2. Eject the tip into the "Used Neon® Tips Box"
   • Write the cell line and DNA/RNA/protein used on the box's lid. Be sure to write your initials, and indicate if the tip was cleaned with DNase
   • The row in which the tip sits indicates its number of uses, and each column should only have one tip in it at a time
3. Allow the tip to air dry for 10 minutes, with the used tips box lid open, in a laminar flow cabinet
4. Be sure to allow at least 12 hours between rinsing in ethanol and re-using the tip, in order to allow all residual ethanol to evaporate. You may also want to rinse a used tip in ddH₂O before using again

Recipes

Replacement electrolytic buffer E2:  

• Add 10% v/v glycerol to a 150 mM solution of NaCl (Final solution is 135 mM NaCl in H₂O + 10% glycerol)

Replacement Buffer "R": There are various options for replacing buffer "R". Because of slight variations from the actual "R" buffer, different cell lines may work optimally in different buffer formulations. Options include:  

• 250 mM sucrose + 1 mM MgCl₂ in 1xPBS
• 250 mM sucrose in 1xPBS
• 125 mM sucrose in 1xPBS
• 150 mM NaCl
• 75 mM NaCl
• 1xPBS
• 0.75xPBS
• 0.5xPBS

DNase I Storage Buffer:  

• 20 mM Tris-HCl (pH 7.5) + 1 mM MgCl₂ in 50% glycerol + H₂O

1.1x DNase I Reaction Buffer (for mixing 9:1 with enzyme stock):  

• 44 mM Tris-HCl (pH 8.0) + 2.75 mM MgSO₄ + 1.1 mM CaCl₂

DNase I Stop Solution  

• 50 mM EDTA in ddH₂O