Gene pulser electroporation buffer reagent

Log growth U937 cells were washed 1 time with PBS and resuspended at 2×10⁷ cells/ml in 1 ml of Gene Pulser electroporation buffer reagent (Bio-Rad, Hercules, CA, USA), mixed with 20 mg of LL-37 RNAi plasmids or mock RNAi plasmids (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Electroporations were performed using a Gene-Pulser (Bio-Rad) at 280 V and 960 mF in 0.4 cm cuvette (Bio-Rad). The samples were transferred to culture flasks containing complete DMEM medium with 10% FCS in 25 cm² and incubated at 37°C in 5% CO₂. After 48 h, the growth medium was changed and puromycin (Invitrogen) was added at a concentration of 5 mg/ml. The culture medium was switched with fresh growth medium (containing 5 mg/ml puromycin), every 4 days. After 4 weeks, positive polyclonal populations (pools) were identified based on western blot analysis for LL-37 expression. Individual positive clones were eventually isolated via limiting dilution analysis in 96-well plates.

Reprogramming of fibroblasts was performed as described before [24 ]. In brief, fibroblasts were detached from cell culture plate and washed with PBS. 4 × 10⁵ cells were resuspended in 400 μl Gene Pulser® Electroporation Buffer Reagent (BioRad) with 23.4 μg of each episomal plasmid (Addgene, Plasdmid #27078, #27080, #27076), containing the reprogramming genes OCT4, SOX2, KLF4, and C-MYC. Cell solution was carefully mixed and electroporated with three pulses of 1.6 kV, capacitance of 3 μF and a resistance of 400 Ω (Gene Pulser II (BioRad)). Fibroblasts were left for recovery in Fibroblast medium without antibiotics, containing 10 μM Rock inhibitor (Y-27632). After cells reached a confluence of 60–70%, medium was changed to TeSR™-E7™ (STEMCELL). Colonies appeared after 21–28 days. These were picked manually and maintained in TeSR™-E8™ (STEMCELL). iPSC lines were characterized for pluripotency [24 ]. Six iPSC lines derived from one individual were selected and used in the present study, three iCTR clones and three RTT Ex3-4 clones.

The viral reprogramming was performed using a lentiviral construct containing the classical reprogramming factors OCT4, SOX2, KLF4 and C-MYC [15 ].
Episomal reprogramming was achieved as described before with small adjustments [16 (link)]. Fibroblasts were dissociated from cell culture plates with Trypsin-EDTA (0,05%) (ThermoFisher). To prepare one well of a 6-well plate, 4 × 10⁵ cells were collected and centrifuged for 5 min at 1200 rpm. Pellet was washed once with PBS and re-suspended in 400 μl Gene Pulser® Electroporation Buffer Reagent (BioRad) containing 23.3 μg of each episomal plasmid (Addgene, Plasmid #27078, #27080, #27076). Cell-plasmid suspension was electroporated in electroporation cuvettes with Gene Pulser II (BioRad). Three pulses of 1.6 kV, with a capacitance of 3 μF and a resistance of 400 Ω were applied. Cells were left to recover overnight in Fibroblast medium without antibiotics but with 10 μM ROCK-inhibitor (Y-27632) in Geltrex®-coated well of a 6 well plate. The next day, medium was changed to Fibroblast medium with antibiotics.
When reprogrammed fibroblasts reached 60–70% confluence, medium was changed to TeSR™-E7™ (STEMCELL) and refreshed daily. Colonies were picked manually after 21–28 days and maintained in TeSR™-E8™ (STEMCELL) on Vitronectin XF™ (STEMCELL) coated 6 well plates [17 ].