P3 primary cell kit

P. falciparum schizont stage parasites were transfected with a 4D-Nucleofector instrument using a P3 Primary Cell kit (Lonza). One day after transfection, drug selection was commenced with 2.5 nM WR99210. Drug selection was applied for four days then removed.

T cells were electroporated with siRNAs against FASN (ThermoFisher assay ID s5031) or Stealth RNAi negative control (medium GC content) siRNA (ThermoFisher) as a non-specific (NS) control using the Amaxa Nucleofection 4D system and the P3 Primary Cell kit (Lonza). Assays were conducted 4 days post-electroporation for peak knockdown efficiency. Knockdown efficiencies were assessed by immunoblotting for every experiment.

Macrophages were transfected with p47^phox specific siRNA (#4392420, Thermo Fisher Scientific) or scrambled control siRNA (SR30004, Origene) using Lipofectamine 2000 (Thermo Fisher Scientific)32 (link). Briefly, MDMs (0.6 × 10⁶/well) were seeded in a 24-well plate. 50 pmol siRNA and 3 μL Lipofectamine 2000 were mixed in 350 μL plain DMEM. The transfection mixture was incubated at room temperature for 30 min. Macrophages were washed with plain DMEM once and then incubated with the transfection mixture at 37 °C. After 2 h, the transfection mixture was removed and replaced with D-10 media. The level of p47^phox knockdown was assessed by RT-PCR and Western blotting 48 h post transfection. TAK-1 specific siRNA (#4390824, Thermo Fisher Scientific) was transfected into macrophages using the 4D-Nucleofector system with the P3 primary cell kit (Lonza)32 (link). M-Mac was transfected with 100 pmol of siRNA following a transfection protocol by the vendor, and then cultured for 48 h. The cells were treated with or without 100 ng/ml of IL-27 for another 48 h and then subjected to western blot for the detection of TAK-1 protein or ROS assay as described above.

ATCC-BXS0116 human iPSCs (ATCC No. ACS-1030) were cultured on Corning Matrigel-coated plasticware in mTESR Plus media (StemCell No. 100-0276) containing 10 μM ROCK inhibitor Y-27632 (StemCell No. 72302) for 24 h before nucleofection. For gene-editing experiments, RNPs were formed as described previously and electroporated into 200,000 iPSCs using the P3 Primary Cell kit (Lonza No. V4XP-3032) in the Lonza 4D nucleofector using the CA-137 program. Immediately before electroporation iPSCs were harvested and dissociated into single cells using Accutase (StemCell No. 07922) according to the manufacturer's protocol. Following electroporation, iPSCs were resuspended in 1 mL of culture medium supplemented with 10 μM ROCK inhibitor Y-27632 and cultured in 24-well plate format for an additional 24 h.
For experiments using AAV-6 transduction, the relevant donor AAV-6 at the indicated MOI was diluted in culture media before electroporation, and added to cells immediately following the electroporation event. Rock inhibitor was removed 24 h postelectroporation. Seven days postelectroporation, cells were harvested for flow cytometry and genomic DNA extraction using Accutase. All primary cells were purchased from commercial organizations with in-house institutional review boards and other safeguards in-place to ensure the ethical sourcing of the material.

Cryopreserved human peripheral blood B cells were purchased from StemCell Technologies (StemCell No. 70023) and expanded using the ImmunoCult™ Human B Cell Expansion Kit according to the manufacturer's protocol for 48 h before nucleofection. For gene-editing experiments, RNPs were formed as described previously and electroporated into 200,000 B cells using the P3 Primary Cell kit (Lonza No. V4XP-3032) in the Lonza 4D nucleofector using the EO-117 program. Following electroporation, B cells were resuspended in 1 mL of culture medium and cultured in 24-well plate format. For experiments using AAV-6 transduction, the relevant donor AAV-6 at the indicated MOI was diluted in culture media before electroporation and added to cells immediately following the electroporation event. Seventy-two hours postelectroporation, cells were harvested for flow cytometry and genomic DNA extraction as described previously.