Cerebral organoids were kept in antibiotics-free conditions before electroporation. Electroporations were performed in 39-day-old cerebral organoids and fixed 7 days after electroporation. During the electroporation, cerebral organoids were placed in an electroporation chamber (Harvard Apparatus, Holliston, MA, USA) under a stereoscope and using a glass microcapillary 1 to 2 μl of plasmid DNAs was injected together with Fast Green (0.1%, Sigma-Aldrich) into different ventricles of the organoids. Plasmid DNA concentrations were as follows: GFP (0.7 μg/μl), miRScr (1 μg/μl), and MIR3607 (1 μg/μl). Cerebral organoids were subsequently electroporated with five pulses applied at 80 V for 50 ms each at intervals of 500 ms (ECM830, Harvard Apparatus). Following electroporation, cerebral organoids were kept for an additional 24 hours in antibiotics-free media, and then changed into the normal media until fixation. Cerebral organoids were fixed using 4% PFA for 1 hour at 4°C, cryopreserved with 30% sucrose, and stored at −20°C. For immunofluorescence, 20-μm cryosections were prepared.
Electroporation chamber
Manufactured by Harvard Apparatus
Sourced in United States
The Electroporation Chamber is a laboratory equipment designed to facilitate the process of electroporation. Electroporation is a technique used to temporarily increase the permeability of cell membranes, allowing the introduction of various molecules, such as DNA, RNA, or drugs, into the cells. The Electroporation Chamber provides a controlled environment for this process, enabling researchers and scientists to perform their experiments effectively.
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9 protocols using electroporation chamber
1
Electroporation of Cerebral Organoids
2
Electroporation of Cerebral Organoids
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Cerebral organoids were kept in antibiotic-free conditions prior to electroporation. Electroporations were performed in cerebral organoids at the 44 days stage after the initial plating of the cells and fixed 7 dpe. During the electroporation cerebral organoids were placed in an electroporation chamber (Harvard Apparatus, Holliston, MA, USA) under a stereoscope and using a glass microcapillary 1–2 μL of plasmid DNAs at final concentration of 1 μg/μL was injected together with Fast Green (0.1%, Sigma) into different ventricles of the organoids. Cerebral organoids were subsequently electroporated with five pulses applied at 80 V for 50 ms each at intervals of 500 ms using the Electroportator ECM830 (Harvard Apparatus). Following electroporation cerebral organoids were kept for additional 24 h in antibiotic-free media, and then changed into the normal media until fixation. Cerebral organoids were fixed using 4% PFA for 1 h at 4°C, cryopreserved with 30% sucrose and then stored in −20°C. For immunofluorescence, 16 μm cryosections were prepared.
3
Electroporation of Cerebral Organoids
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For electroporation (see scheme in Fig. 6c ), cerebral organoids were kept in NDM + A without antibiotics. The organoids were placed in an electroporation chamber (Harvard Apparatus) and pCMV-SPORT6 plasmid with FICD wt, FICD E234G (gift from A. Itzen, TUM), or FICD H363A plus pCAG-IRES-GFP (FICD to GFP ratio 2:1), GFP only as overexpression control, miRNA against FICD (or scrambled miRNA negative control) in pCAG-GS at a concentration of 1 µg/µl, supplemented with fast green for visualisation, was injected into ventricle-like cavities at several positions per organoid. Electroporation was performed with an ECM830 electroporation device (Harvard Apparatus) by subjecting the organoids to a 1 s interval with 5 pulses of 50 ms duration at 80 mV.
4
Brain Organoid Electroporation Protocol
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Human brain organoids were kept in neural differentiation medium without antibiotics for 2 h before the experiment. The organoids were then placed in an electroporation chamber (Harvard Apparatus, Holliston, MA, USA), and the plasmid DNA was injected at a concentration of 1 μg/μl in several positions (Cárdenas et al, 2018 ; Klaus et al, 2019 ). The organoids were then subjected to five pulses at 80 V with a 50‐ms duration in an interval of 500 ms using an ECM830 electroporation device (Harvard Apparatus).
5
Electroporation of Chick Organoids
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COs were kept in antibiotic-free conditions prior to electroporation. Electroporation was performed in COs at around 40d stages after the initial plating of the cells, and fixed 4dpe. During electroporation, COs were placed in an electroporation chamber (Harvard Apparatus, Holliston, MA, USA) under a stereoscope, and using a glass microcapillary of 1–2 µL, plasmid DNA was injected together with Fast Green (0.1%, Sigma) into different ventricles of the COs. COs were subsequently electroporated with 5 pulses applied at 80 V for 50 ms each, at 500 ms intervals (ECM830, Harvard Apparatus). Following electroporation, COs were kept for additional 24 h in antibiotics-free media and then changed into the normal media until fixation. COs were fixed using 4% PFA for 1 h at 4 °C, cryopreserved with 30% sucrose and stored at −20 °C. For immunofluorescence, 16 µm cryosections were prepared. For each experiment, many independent ventricles per CO from at least 3 different COs generated in 3 independent batches were analyzed.
6
Cerebral Organoid Electroporation Technique
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Cerebral organoids were kept in antibiotics-free conditions prior to electroporation. Electroporations were performed in cerebral organoids at 39 days stages after the initial plating of the cells and fixed 7 days post electroporation. During the electroporation cerebral organoids were placed in an electroporation chamber (Harvard Apparatus, Holliston, MA, USA) under a stereoscope and using a glass microcapillary 1-2 μL of plasmid DNAs was injected together with Fast Green (0.1%, Sigma) into different ventricles of the organoids. The plasmid DNAs injected were a mix of 0.75 mg/ml GFP with or without 1mg/ml myr-Robo1, 1mg/ml myr-Robo2, 1mg/ml Dll1 gRNA (+Cas9). Cerebral organoids were subsequently electroporated with 5 pulses applied at 80V for 50ms each at intervals of 500ms (ECM830, Harvard Apparatus). Following electroporation, cerebral organoids were kept for additional 24hr in antibiotics-free media, and then changed into the normal media until fixation. Cerebral organoids were fixed using 4% PFA for 1hr at 4°C, cryopreserved with 30% sucrose and stored at −20°C. For immunofluorescence, 16 μm cryosections were prepared.
7
Electroporation of Cerebral Organoids
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COs were electroporated at two different time points: at day 20 and day 35 after plating the iPSCs on the 96-well plate. For electroporation, COs were kept in NDM+A medium without Antibiotic Antimycotic solution and moved to an electroporation chamber (Harvard Apparatus). Using a stereoscope to localize the ventricle-like cavity (VL), 1–2 μl of each plasmid (pCAGGS-GFP, pCAGGS-GFP-IRES-GNG5, 2/3 of pCAGGS-GFP + 1/3 of pCAGGS-GAP43-GFP or 2/3 of pCAGGS-GFP-IRES-GNG5 + 1/3 of pCAGGS-GAP43-GFP) to a final concentration of 1 μg/μl mixed with 0.1% Fast-Green (F7252, Sigma Aldrich) were injected using Glass Micropipettes (5-000-1001-X10, Drummond Scientific) and electroporated with five pulses applied at 80 mV for 50 ms each at intervals of 500 ms (ECM830, Harvard Apparatus). 24 hours after electroporation COs were moved to new NDM+A medium and kept in culture for 7 additional days until they were fixed for 2 h in 4% PFA. After fixation, COs were transferred to 30% sucrose in PBS overnight for cryopreservation, embedded in OCT Compound (361603E, VWR Chemicals) and stored at −20°C.
For immunohistochemistry, 14 μm sections were prepared with a cryostat. For each analysis, at least 3 different COs per condition were analyzed from 2 independent batches.
For immunohistochemistry, 14 μm sections were prepared with a cryostat. For each analysis, at least 3 different COs per condition were analyzed from 2 independent batches.
8
Transfection of aRG in Cerebral Organoids
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For transfection of aRG in germinal zones of COs by electroporation (see scheme in Fig 1 D), COs were kept in NDM + A without antibiotics and antimycotics. The organoids were placed in an electroporation chamber (Harvard Apparatus), and miRECE2 or miRneg expression plasmid (containing GFP) at a concentration of 1 μg/μl, or ECE2‐OX plasmid in 2:1 ratio with pCAG‐IRES‐GFP (total concentration of 1 μg/μl) vs. pCAG‐IRES‐GFP as control, always supplemented with Fast Green (0.1%; Sigma) for visualisation, was injected into ventricle‐like cavities at several positions per organoid. Electroporation was performed with an ECM830 electroporation device (Harvard Apparatus) by subjecting the organoids to a 1‐second interval with five pulses of 50‐ms duration at 80 mV. Medium was changed to antibiotic‐containing NDM + A on the next day.
9
Cerebral organoid electroporation protocol
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Cerebral organoids were kept in antibiotics‐free conditions prior to electroporation. Electroporations were performed in cerebral organoids at 40 days stages after the initial plating of the cells and fixed 7 days post‐electroporation. During the electroporation, cerebral organoids were placed in an electroporation chamber (Harvard Apparatus, Holliston, MA, USA) under a stereoscope and using a glass microcapillary 1–2 μl of plasmid DNAs was injected together with Fast Green (0.1%, Sigma) into different ventricles of the organoids. Plasmid DNA concentrations were as follows: Gfp (0.7 μg/μl), Irs2 (1 μg/μl), TUD‐Scr (1 μg/μl), and TUD‐let7 (1 μg/μl). Cerebral organoids were subsequently electroporated with five pulses applied at 80V for 50 ms each at intervals of 500 ms (ECM830, Harvard Apparatus). Following electroporation, cerebral organoids were kept for additional 24 h in antibiotics‐free media and then changed into the normal media until fixation. Cerebral organoids were fixed using 4% PFA for 20 min at 4°C, cryopreserved with 30% sucrose, and stored at −20°C. For immunofluorescence, 20‐μm cryosections were prepared.
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