Gene pulser xcell device

Before electroporation, 20 μl of the DNA solution was placed on a Millipore VSWP02500 membrane (25 mm diameter, 0.025 μm porosity) floating on a Petri dish filled with 40 ml of ultrapure sterile water. After 1 hour of dialysis, the droplet is recovered by pipetting. 1 μl of dialyzed DNA was mixed with 50μl electro-competent E. coli Top 10 cells in pre-chilled 1 mm electrodes distance Gene Pulser cuvette (Biorad) and the electroporation was then performed with Gene Pulser Xcell device (voltage 1800V, capacitance 25μF, resistance 200 ohm). Electroporated cells were recovered with 1ml of LB and incubated at 37 C with orbital shaking during 45 minutes before plating on LB plates containing the appropriate antibiotics. The number of transformants is estimated by plating serial 10-fold dilutions and counting colony forming units (cfu) after overnight incubation at 37 C. Typically, between 10⁶ and 10⁷ cfu were obtained per electroporation. Alternatively, the DNA was concentrated at least 10-fold using the “DNA Clean & Concentrator^™-5” kit from Zymo Research and up to 10⁸ cfu can be obtained with 5 μl in a single electroporation.

Plasmid DNA was extracted using the EZ-10 Spin Column Plasmid DNA Minipreps kit (Bio Basic) following the manufacturer’s instructions. Enzymes used in this study were purchased from New England Biolabs. PCR assays were performed with the primers listed in Table 2. PCR conditions were as follows: (i) 3 min at 94°C; (ii) 30 cycles, with 1 cycle consisting of 30 s at 94°C, 30 s at the appropriate annealing temperature, and 1 min/kb at 68°C; and (iii) 5 min at 68°C. When required, the resulting products were purified using the EZ-10 Spin Column PCR Products purification kit (Bio Basic) following the manufacturer’s instructions. E. coli strains were transformed by electroporation as described previously (64 (link)) in a Bio-Rad Gene Pulser Xcell device set at 25 μF, 200 V, and 1.8 kV using 1-mm gap electroporation cuvettes.

Mature CBDC were loaded with WT1-encoding mRNA (AmpTec GmbH) by electroporation (EP) as previously described (24 (link)), with minor modifications. Briefly, 5–10×10e6 cells in 200 µl OptiMEM media were transferred to a 4-mm electroporation cuvette (Bio-Rad, Hercules, CA, USA) and electroporated with 9,5 ug RNA by a time constant pulse of 300 V for 7 ms using the Gene Pulser Xcell device (Bio-Rad). After EP the cells recovered for 4h in the medium used to mature the cells. Next, WT1 expression was determined. Cells were washed with FACS buffer (PBS containing 2% BSA (Sigma-Aldrich) and 0.1% sodium azide (NaN3, Sigma-Aldrich) prior to a 15 min incubation at room temperature (RT) in the dark with a fixable viability dye (Thermo fisher). Next, cells were washed and fixed/permeabilized (eBioscience) for 30 min at 4°C. After washing with permeabilization buffer, anti-WT1 (F6-H2; (Dako) or purified mouse IgG1κ (MG1-45; Biolegend) as isotype control and FcR Blocking (Miltenyi Biotec) was added for 15 min at RT in de dark. The cells were washed again with permeabilization buffer before adding the F(ab’)2 Anti-mouse IgG PE antibody (eBioscience) for 15 min at RT in the dark. Cells are resuspended in FACS buffer and analyzed using a FACS CantoII (BD) flow cytometer. Analysis was performed using FlowJo software (Tree Star, Inc.).