Neon pipette tip

Human MDMs were electroporated with PBS, mouse anti-GFP (9F9.F9; Abcam) or mouse anti-NLRP3 (Cryo-2; Adipogen) IgG antibodies. All antibodies used for electroporation were passed through Amicon Ultra-0.5 100 kDa centrifugal filter devices (Millipore) to remove traces of azide and replace buffer with PBS. All antibodies were diluted to 0.6 mg/mL in PBS prior to electroporation. Antibody electroporation was performed using the Neon Transfection System (Thermo Fisher Scientific). MDMs were washed with PBS and resuspended in Buffer R (Thermo Fisher Scientific) at a concentration of 1.4 × 10⁸ cells/mL. For each electroporation reaction 1.4 × 10⁶ cells (10 μl) were mixed with 2 μl of antibody or PBS. The mixture was taken up into a 10 μl Neon® Pipette Tip (Thermo Fisher Scientific) and electroporated using the following settings: 1400V, 20 ms, 2 pulses. Electroporated cells were transferred to growth medium without antibiotics.

Transfections of hiBC line No4 were performed by electroporation using the Neon Transfection System (Thermo Fisher Scientific, USA). Cells were harvested with the Versene solution and re-suspended at a concentration of 3×10³ cells/μL in Opti-MEM (Thermo Fisher Scientific, USA) with 100× GlutaMAX (Thermo Fisher Scientific, USA) and 10 μM Y-27632 and 0.2 μg pEGFP-С1 plasmid (Clontech, USA) per 3×10⁴ cells. The cell suspension with plasmid was loaded into a 10 µL Neon Pipette Tip (Thermo Fisher Scientific, USA) and electroporated with two consecutive pulses at 1290 V for durations of 20 msec. Then, cells were placed into a culture dish with BCM with 10% fetal bovine serum (FBS) and 10 μM Y-27632 (without antibiotics). After 24 h, the medium was replaced with BCM without 10% FBS and 10 μM Y-27632 (with antibiotics). The visualization of the cell fluorescence was carried out using the Lionheart FX Automated Microscope. Transfection efficiencies were assessed 48 h after infection by flow cytometry using a CytoFLEX S (Beckman Coulter, USA).

Electroporation of full-length KRas constructs (KRasWT, KRasG13C, KRasG13C-edaGDP, KRasG13C:acetyledaGDP and KRasG13C-edaGppCp) was performed based on the protocol described by Alex et al., 2019 (link) using the Neon Transfection System Kit (Thermo Fisher). For electroporation, 3 million cells per experiment were harvested by trypsinization, washed with PBS, and resuspended in 85 µL of the electroporation buffer R (Thermo Fisher). Increasing amounts of recombinant protein samples for each construct (0, 50, 100, 200 and 300 µg) were diluted 1:1 in buffer R followed by the addition of 30 µL of this protein master mix to the cell suspension. This cellular slurry was loaded into a 100 mL Neon Pipette Tip (Thermo Fisher) and electroporated with 2x35 ms pulses at 1000 V. After electroporation, the cells were washed twice with PBS (15 mL) to remove non-internalized extracellular protein and the cell pellet was resuspended in 2 mL complete growth media. Cells were transferred into six-well tissue culture plates (Sarstedt) and incubated for 24 hr at 37 °C and 5% CO₂ in a humidified incubator for recovery before being processed for western blotting analysis.