Plasmid nucleofection

Electroporation of organoids using the Lonza Nucleofection system.

Before you start

• Pre-warm TrypLE enzyme to 37°C.
• Warm P3 Nucleofection Buffer to room temperature before use.

Things you will need

Pipettes and filter tips (p1000, p200, p20)

1.5 ml tubes

15 ml Falcon Tubes

TrypLE Express Enzyme 1X (ThermoFisher Scientific 12605010)

30 μm cell strainers (CellTrics® filters 30um, Sysmex, 04-004-2326)

24 well plates (Greiner Bio One cat.no. 665980)

20 μl Nucleocuvette Strip and P3 Nucleofection Buffer  from P3 Primary Cell 4D-Nucleofector kit (Lonza, cat. V4XP-3032, 32 reactions) 

Advanced DMEM/F12 +++ (protocol 4.1) for washing.

Matrigel, Basement Membrane Matrix, Growth Factor Reduced (GFR), Phenol Red-free (BD cat.no. 356231)

Human embryonic lung self-renewal growth medium (protocol 4.2)

Human embryonic lung self-renewal growth medium (protocol 4.2) + 10 μM Rho Kinase inhibitor Y27632 (Sigma-Aldrich, Y0503)

Nucleofection of organoids

1. Culture organoids to reach 80-90% confluence.
2. Remove the medium from the organoid culture. 
3. Wash the well briefly with 1x PBS. 
4. Remove the PBS and add 500 μl of pre-warmed TrypLE express enzyme.
5. Triturate (pipette up and down 10 times) with P1000 pipette to break up the organoids and incubate at 37°C for 5 minutes.
6. Triturate by pipetting up and down with P200 pipette about 20 times.
7. Check that single cells have been obtained (if not, incubate a further 2-5 minutes at 37°C) and add 1 ml cold Advanced DMEM/F12 +++ to stop the reaction.
8. Filter through a 30 μm cell strainer.
9. Wash cell strainer with 2 ml of Advanced DMEM/F12 +++  and count the cell number.
10. Prepare aliquots of  ~200,000 cells for each transduction. Pellet cells at 200-500 rcf for 5 minutes and resuspend in 20 μl P3 Nucleofection Buffer.

Preparation of 20 μl of P3 nucleofection buffer: 16.4 μl Nucleofector solution and 3.6 μl of supplement.

1. Transfer the cells to the nucleocuvette strip and, working quickly, nucleofect the cells using a suitable programme.

Several nucleofection programmes should be tested to select the on which is most efficient for the organoids used.

1. Dilute P3 nucleofection buffer using 200 μl human embryonic lung self-renewal growth medium + 10 μM Rho Kinase inhibitor (Y27632).
2. Remove cell suspension from the cuvette into a 1.5 ml tube. 
3. Spin cells at 200-500 rcf, 5 minutes. Discard supernatant and resuspend in 100 μl of matrigel.
4. Seed into two wells of a pre-warmed 24 well plate and incubate at 37°C 10-15 minutes to allow matrigel to solidify. Add 600 μl human embryonic lung self-renewal growth medium + 10 μM Rho Kinase inhibitor (Y27632). 
5. After 2-3 days change the medium to human embryonic lung self-renewal growth medium.

Media protocols

 

1. Advanced DMEM/F12 +++ medium

FGF10 (100 µg/ml)                            20 µl

FGF7 (50 µg/ml)                                40 µl

CHIR99021 (10 mM)                         6 µl

SB43152 (10 mM)                              20 µl