Amaxa rat nsc nucleofector kit

Prior to nucleofection, cells were harvested with trypsin; 5x10⁶ cells were nucleofected using the Amaxa Rat NSC Nucleofector Kit (Lonza) according to the manufacturer protocol. NSC were resuspended in 100 μL of nucleofection solution with 5 μg of DNA and 0.5 μg of pmaxGFP Vector or 1.5 μg (1 μM) of siRNA and electroporated with the Amaxa Nucleofector Program A-033 (Lonza) before plating on poly-ornithine/laminin coated dishes. For shRNA nucleofection, five million NSC resuspended in 100 μL of nucleofection solution of the Amaxa^® Rat NSC Nucleofector^® Kit (Lonza) with 5 μg of shRNA (1 μM) were electroporated with the Amaxa Nucleofector Program A-033 (Lonza) before plating on poly-ornithine/laminin coated dishes. All cells were incubated in humidified 37°C/5% CO₂ incubator and medium was changed after 24 hr.

ITSCs were cultivated under standard conditions, digested with collagenase NB4 (SERVA Electrophoresis) and harvested via centrifugation. Afterwards, ITSCs were transfected with 1 μg pmax GFP (Amaxa Biosystems, Lonza Group AG, Basel, Switzerland) using Amaxa rat NSC-Nucleofector Kit (Amaxa Biosystems) and Nucleofector II device (Amaxa Biosystems) according to the manufacturer’s guidelines.
Immediate addition of 5 ml DMEM/F-12 (Sigma-Aldrich) was followed by centrifugation for 10 minutes at 300 × g. Transfected ITSCs were suspended in 2 ml standard medium supplemented with 10% BP and injected into Afc-FEP bags 2PF-0002 (Afc) using a syringe (B. Braun Melsungen AG). Cultivation was performed at 37°C, 5% CO₂ and 5% O₂ for 48 hours in a humidified incubator (Binder). Live imaging of GFP-ITSCs was done via optical sectioning (Z-Stack) using confocal laser scanning microscopy (excitation wavelength: 488 nm, LSM 510, Carl Zeiss, Jena, Germany), while ZEN software (Carl Zeiss) was subsequently applied for three-dimensional reconstruction.