Wash T cells (from Basic Protocol 1 or Basic Protocol 2) in PBS to remove tracesof FBS, which can contain RNAses.
3. Suspend T cells at 2 × 106 cells per 20 µl nucleofection buffer (included in the
Primary Cell 4D-Nucleofector X Kits).
A wide range of cell numbers (between 105 to 107 cells per nucleofection) can be used
without loss in knockout efficiency or viability.
For human T cells: P2 nucleofection buffer.
For mouse T cells: P4 nucleofection buffer.
T cells should be suspended in nucleofection buffer immediately before nucleofection, as
the nucleofection buffer is toxic to cells.
Work carefully but as quickly as possible from steps 3 to 11 to minimize the amount of
time the T cells are in the nucleofection buffer.
4. Add 5 µl of RNP (obtained following Basic Protocol 3), and mix by repeated
If using a pool of RNPs (we have tested up to 6 RNPs), reduce the volume of each RNP
to 3 µl. The entire volume of the cell/RNP mix is then used in subsequent steps.
5. Incubate 2 min at room temperature.
6. During the incubation, turn on the 4D-Nucleofector machine.
7. Choose the correct preset program for electroporation.
a. For human T cells: Primary Cell P2.
b. For mouse T cells: Primary Cell P4
8. Enter the appropriate pulse code.
a. For human T cells: EH100.
b. For mouse T cells: DS137 for resting cells; and CM137 for activated cells.
9. Transfer the T cell/RNP mix to Nucleocuvette strips.
Pipet gently to avoid air bubbles.
10. Place cuvette strips in the 4D-Nucleofector machine, and electroporate using the
program and pulse code selected in steps 7 and 8.
11. Remove cuvette strips from the 4D-Nucleofector machine and the 96-well plate with
pre-warmed medium from the 37°C incubator. Immediately add 100 µl pre-warmed
T cell medium to a nucleofection cell, mix gently by pipetting once or twice, and
transfer the entire volume (25 µl T cells and RNPs plus 100 µl T cell medium) from
the nucleofection cell to the remaining 100 µl of T cell medium in the appropriate
well of the 96-well plate.
When using more than 2 × 106 cells per transfection, T cells should be cultured in
multiple wells at a max. of 2 × 106 cells per well.
12. Incubate the 96-well plate containing the electroporated cells at 37°C incubator.
Allow electroporated T cells to recover for 2 hr before any further manipulation.
Detailed method paper using Nucleofector 4D to transfect CRISPR Cas9 RNPs into non stimulated (and stimulated) mouse and human T cells.
Showing optimization of RNPs quantity in a Nucleofection experiment and the use of an enhancer (from IDT) to reduce RNPs amounts. The authors identified P2 / EH-100 as optimal Nucleofection conditions for human T cells (stimulated and non-stimulated), P4 / DS-137 for the mouse unstimulated T cells and P4 / CM-137 for the mouse stimulated T cells.
Earlier work from the same last author presented this approach in a research paper (Seki, et al. J Exp Med. 2018)