Amaxa se cell line 4d nucleofector x kit

For cell-based apoptosis assay, the wild type (WT) and F122A, I128D mutants of full length Caspase-8 (residues 1–479) and DEDs (residues1–209) were subcloned into pEGFP-N1 vector. HeLa cells (3 × 10⁵) were transfected with plasmids (4 μg) as indicated in figure legends using TransIn EL Transfection Reagent (TransGen Biotech, China). For Jurkat cells, 2 μg plasmids were electroporated into 1 × 10⁶ cells with Amaxa SE Cell Line 4D-Nucleofector X Kit (Lonza Group Ltd, Basel, Switzerland). 18 hr post transfection/electroporation, cells were further stimulated with anti-Fas (1 μg/ml) for 20 hr before harvest. The cell sample were then analyzed for apoptosis via Annexin V staining (eBioscience) using FACSCalibur flow cytometer (Becton Dickinson, NJ, USA). Apoptosis rates were quantified as the percentage of Annexin V-APC positive versus GFP positive cells.

Nrp2-deficient MC3T3-E1 cells were generated by nucleofection of CRISPR/Cas9 ribonucleoprotein complexes (IDT, Leuven, Belgium) using a 4D nucleofector system (Lonza, Cologne, Germany) and the Amaxa SE Cell Line 4D-Nucleofector X Kit (catalog-no. V4XC1032, Lonza) according to the manufacturer’s instructions. Resulting clones were checked by genotyping, mRNA and protein expression for the absence of Nrp2 (Supplementary Methods and Data).