Electroporation of CD34+ cells

Protocol for electroporating CD34+ cells with ABE 8e mRNA using the MaxCyte system

Materials and Reagents:

1 million 48 hours expanded CD34+ cells

HSC/CD34+ expansion media (components described in materials and methods “Erythroid differentiation”)

ABE 8e mRNA (5 µg)

Desired gRNA 100 pmole (Synthego)

MaxCyte buffer (Hyclone)

OC25 × 3 Maxcyte cuvettes

12-well plate (Corning)

Procedure:

• Expand CD34+ cells in HSC/CD34+ expansion media for 48 hours.
• Pellet around 1 million CD34+ cells.
• Resuspend the pellet in 19 µl of MaxCyte buffer (Hyclone).
• Add 5 µg of ABE 8e mRNA (5 µl) and 100 pmole of the desired gRNA (1 µl) from Synthego to the cell suspension. Mix thoroughly.
• Load the 25 µl cocktail, as prepared above, into an individual well of an OC25 × 3 Maxcyte cuvette. Given that the experiments will be conducted in triplicate, each cuvette will be dedicated to a single sample.
• Electroporate the cells using program 'HSC-3'.
• After electroporation, transfer the contents of the cuvette to a single well of a 12-well plate (Corning).
• Allow the cells to recover for 20 minutes in an incubator at 37°C with 5% CO₂.
• After the recovery period, add 1 ml of HSC expansion media to the cells in the 12-well plate.
• Expand the cells in the 12-well plate for 48 hours in an incubator at 37°C with 5% CO₂ before performing any further experiments.