If T-cell isolations are performed on Monday or Tuesday, nucleofections can be performed
on Thursday or Friday of the same week, allowing 72 h for T-cell stimulation.
48 Prepare the stimulatory beads from the T Cell Activation and Expansion Kit according to the
manufacturer’s instructions. Allow the beads to rotate at 4 °C for at least 2 h before use.
49 Mix the P3 nucleofection buffer with the required supplement from the P3 Primary Cell 4DNucleofector
X Kit according to the manufacturer’s instructions. Allow the supplemented
nucleofection buffer to come to room temperature before it comes into contact with the cells.

CRITICAL STEP! Supplemented nucleofection buffer is stable at 4 °C for up to 3 months. Do not
prepare more supplemented nucleofection buffer than will be used in this time frame.
50 Set up the Amaxa 4D Nucleofection System according to the manufacturer’s instructions, specifying
buffer P3 and pulse code EH-115.
51 Prepare cRPMI plus 20 U/mL IL-2 and pre-warm it to 37 °C.
52 Determine the concentration of activated CD4+ T cells from Step 45, using a hemocytometer or
automated cell counter. Each nucleofection reaction will require 500,000 cells per well. Calculate the
total number of cells required from each donor, remove the required volume of culture and place it
into a 50-mL conical tube, and pellet the cells by centrifugation in a spinning bucket rotor at 400g
for 5 min at room temperature. For example, pellet 2 million cells for four nucleofection reactions.

CRITICAL STEP! This procedure is effective when using anywhere between 200,000 and 1 million
cells per reaction. The cell number used per reaction should be optimized on the basis of the
required cell numbers in downstream applications.
53 Thaw crRNPs in plate format from Step 17 or, if crRNPs are not in plate format, array 3.5 µL of
each crRNP in a 96-well, LoBind, V-Bottom plate in the exact layout to be used for nucleofection.
Bring to room temperature.
CRITICAL STEP Supplemented nucleofection buffer is toxic to cells. To minimize the amount of
time the cells spend in supplemented nucleofection buffer, collect and arrange the necessary
reagents beforehand for efficient handling.
54 Remove the supernatant from the cell pellet obtained in Step 52 and suspend the cells in 20 µL of
supplemented nucleofection buffer per reaction. If working in plate format, transfer the cell
suspension to an appropriate vessel for multichannel pipetting.
55 For each nucleofection reaction, add 20 µL of the cell suspension to 3.5 µL of crRNPs as laid out in
Step 53. Mix gently three to four times by pipette. If working in plate format, this can be done using
a multichannel pipette.
56 Immediately transfer 20-µL aliquots of the cell/crRNP mixture to the nucleofection strip tubes/
plate, leaving any extra solution behind. To avoid arc errors during nucleofection, dispense the
reaction at the bottom of the wells and avoid the formation of air bubbles.
CRITICAL STEP Although arc errors will not prevent the nucleofection from being successful, they may negatively impact editing efficiency and/or cell viability

57 Tap the nucleofection strip tubes/plate gently on the surface of the hood to release any air bubbles that may have formed.
58 Nucleofect the cells on the Amaxa 4D-Nucleofector System, using program EH-115 as set up
in Step 50.
59 As soon as possible after nucleofection, add 80 µL of pre-warmed cRPMI plus 20 U/mL IL-2 from
Step 51 to each well of the nucleofection strip tube/plate. Afterward, move the nucleofection strip
tubes/plate to the sterile tissue culture incubator (37 °C with 5% CO2) for at least 15 min to allow
for cell recovery.
60 As the cells recover, prepare a flat-bottom, 96-well plate containing 100 µL of pre-warmed cRPMI
plus 20 U/mL IL-2 from Step 51 plus T Cell Stimulation Beads. Sufficient beads should be added to
achieve a 1:1 bead/cell ratio in accordance with the manufacturer’s instructions. Store this plate in
the 37 °C incubator to keep it warm until needed.
61 After allowing sufficient time to recover, transfer the entirety of each reaction mixture from the
nucleofection cuvette to the appropriate wells of the pre-warmed flat-bottom plate (from Step 60)
for expansion in culture.

Lonza summary:

Detailed method paper using Nucleofector 4D - X unit (+ Shuttle) to transfect (multiplex) CRISPR Cas9 RNPs into stimulated human T cells to block HIV-host factors, for high-throughput identification and validation studies of HIV drugable targets. Earlier work of the same first author presented the approach in a research paper (Hultquist, et al. Cell Rep. 2016)