Neon transfection of primary mouse T cells

Day 0:

Activate CD4+ T cells O/N on plate-bound stimulation.

Day 1:

1. Pre-coat new plate with goat anti-hamster antibody (24 well for

2. Prepare tubes with RNA for transfection (1ul of 5uM prep per transfection)

  • Set up Neon machine at this point.

3. Prepare plate- wash twice with PBS and add 1x anti-CD3 and -CD28 media with NO ANTIBIOTICS

  • 500 ul per well for 24-well plate and 1ml per well for 12-well plate
  • place in incubator while setting up the rest of the transfection

4. Remove activated cells from plate-bound stimulation to 15ml falcon tube

  • Combine cells of same condition/genotype

5. Count cells (take appropriate number of cells for 200k-400k per transfection)

6. Spin cells at 1200rpm for 5'.

7. Resuspend each set of cells in 1ml PBS and transfer to 1.5ml eppendorf tube.

8. Spin in tabletop centrifuge at 400xg for 5' -> Try to aspirate all PBS without disturbing pellet after spin.

9. Resuspend in 10.5ul R buffer per transfection for that set of cells

  • i.e. if you are going to transfect 5 different miRNAs you would have 1-2 million cells, and you would resuspend them in ~52.5ul R buffer (you can round up a little to avoid running out of cells on the last transfection).

10. Transfer 10.5ul of cells (per transfection) to the tube containing RNA

  • I use 10.5ul instead of 10ul to avoid bubbles in the neon transfection tip.
  • At this point I have a set of tubes with cells and appropriate RNA combined.

11. Using neon pipette and tip, transfect ~10ul of cell/RNA mixture

  • For day 1 cells, I set the machine to: 1550 V, 10mS, 3 pulses
  • Expel transfected cells into previously prepared (antibody coated) well with media.

12. Repeat as necessary for all transfections to be done

  • Can repeat exact same transfection and combine cells in the same well if more than 300k cells are needed for a given condition

* Note: I prepare a separate eppendorf or 96 well with 200+ ul of sterile PBS to "wash" tip before transfecting new set of cells and/or different miRNAs. I pipette up and down 4 or 5 times in the PBS to "wash". I have used one tip up to 12 times, if you have more than 12 transfections I would change tips at some point in the middle.

Can transfect cells again on later days in same manner (on day 4 it appears the voltage can be reduced to 1500V and is still optimal for transfection efficiency with less cell death).