Nucleofection (Timing: 30 min)
In the process of introducing RNP complexes into NK cells, we employ Nucleofection with the Lonza 4D Nucleofector. Here, we delineate the optimized Nucleofection programs we have established and offer explicit instructions on using this Nucleofector device.
43. Prepare the primary NK cell or NK-92 culture medium by warming it up (needed in step 46).
44. Increase efficiency and save time by pre-programming the Nucleofector device (Lonza). Preselect the well/cuvette positions you will use. Ensure to specify the buffer that is used to resuspend the cells before choosing the Nucleofection program.
a. Use program CA-138 for primary human resting NK cells.
b. Use program CM-137 for NK-92 cells.
45. Resuspend the washed and pelleted NK cells in the P3 solution containing the RNP complexes. Pipet carefully but thoroughly. Do not make bubbles.
Note: Cells should not be resuspended in P3 solution for longer than 15 min. It is recommended to perform Nucleofection in multiple batches if you have many samples, depending on the speed of your pipetting work.
46. Immediately transfer the cell/RNP complex mixture to the Nucleofector strip or cuvette.

47. Gently tap the strip/cuvette. Make sure the mixture is uniformly distributed at the bottom of the strip/cuvette and that no bubbles are trapped between the electrodes.
Note: Bubbles trapped between the electrodes can also be removed using a 20 mL pipette tip by pushing them aside.
48. Place the Nucleofector strip or cuvette in the Nucleofector device. Press ‘start’.
49. Take out the Nucleofector strip or cuvette and transfer back to a sterile environment.
50. Add pre-warmed culture medium (supplemented alpha-MEM for NK-92 cells; supplemented RPMI for primary NK cells) in each well immediately after Nucleofection without resuspending the cells. Do not remove the cells from the strip/cuvette yet.
a. Add 1 mL in a cuvette.
b. Add 125 µL to strips.
51. Let the cells recover in an incubator (37°C, 5% CO2) for 15 min.
52. Transfer the cells to plates or flasks.
a. Culture the primary NK cells at 1 3 10^6 cells/mL in a T25 bottle, 6-well plate, or 24-well plate, depending on the number of CRISPR-edited cells.
Note: When keeping the primary NK cells in culture for longer periods of time, addition of low amounts of cytokines are needed to ensure proliferation and survival (i.e., IL-2 (100 U/mL) and IL-15 (5 ng/mL), added every three to four days).
b. Culture the NK-92 cells at 0.5 3 10^6 cells/mL in a T25 bottle, 6-well plate, or 24-well plate, depending on the number of CRISPR-edited cells.
i. Add 100 U/mL IL-2 to the NK-92 cells.