Jacobs:Protocol Neon Transfection

From OpenWetWare

Jump to: navigation, search

Neon Transfection

Materials

  • Cell culture plates or dishes
  • Growth media +/- antibiotics
  • PBS without ions
  • Trypsin
  • Microcentrifuge tubes
  • Neon Transfection Kit (buffers, tips, tube)
  • Kam Lab: Neon pipette & system

Procedures

  1. Count cells. Instead of resuspending the cell pellet in 5ml media, resuspend in PBS.
  2. Prepare your plates with media without antibiotics.
  3. If necessary, prepare the necessary concentration of siRNA.
    • Concentration of siRNA is the concentration in the well with media. Optimized concentration for MC3T3s is 100nM and for hMSCS is 30nM. For all other cell lines, optimize by trying the transfection at 10, 30, 100, and 200 nM of BLOCK-iT for electroporation (Invitrogen #13750062).
    • Math here is more complicated. You need to take into consideration how much of the transfected suspension you add per well and what concentration of siRNA needs to be in that amount. The addition of siRNA should be small enough to be negligible in changing the suspension volume (and cell concentration).
  4. Centrifuge again and resuspend the cell pellet in Resuspension Buffer R.# Your final density should be 1x10⁷ cells/ml.
    • ul of buffer = (# of cells) * (10³ ul / 1x10⁷ cells)
    • Simplified: ul of buffer = (# of cells in thousands)/(10)
    • Example: 500,000 cells -> 500/10 = 50 ul of buffer
  5. Transfer cell suspension to a microcentrifuge tube. Add siRNA and gently pulse.
  6. Bring the plates and cell suspension to the Kam lab along with 1000ul pipette & tips for the Electrolytic Buffer and an appropriate pipette and tips for seeding.
  7. Setup a Neon Tube with 3 mL Electrolytic Buffer. Use Buffer E for the 10 ul Neon Tip and Buffer E2 for the 100 ul Neon Tip.
  8. Set the desired pulse conditions on the device.
    • For MC3T3s, use the Saos-2 cell protocol from Invitrogen.
      • Pulse voltage: 1,200 v
      • Pulse width: 40 ms
      • Pulse number: 1
    • For hMSCs, use the hMSC protocol from Invitrogen.
      • Pulse voltage: 990 v
      • Pulse width: 40 ms
      • Pulse number: 1
    • For all other cells, find a similar cell line on the Invitrogen database and go from there.
  9. Insert the Neon Tip into the Neon Pipette.
    • Make sure to press the push-button to the second stop to open the clamp allowing the clamp to pick up the mounting stem of the piston.
    • Release the push-button but continue to apply downward pressure on the piepette. Make sure the tip is sealed onto the pipette without any gaps.
  10. Pipet the cell suspension using the Neon Piepette to the first stop. Avoid air bubbles during pipetting. This can cause arcing during the electroporation leading to lowered or failed transfection.
  11. Insert the Neon Pipette with the sample into the Neon Tube / Pipette Station until you hear a click sound.
  12. Press start. Complete is displayed on the screen to indicate that electroporation is complete.
    • If your pipette tip has a bubble, the system should show an error and ask you to try again. Otherwise, arcing may occur within the suspension causing a spark and killing your cells.
  13. Remove the Neon pipette and transfer the sample by pressing to the first stop. You can use the Neon Tip about 5 times. Press the push-button to the second stop to remove the tip.
    • If you are not using the full 10 ul in each well or dish, transfer to a microcentrifuge tube and pipette the appropriate amount to the well or dish.
  14. Plate the cells in antibiotic-free media for 4-6 hours.
Personal tools