Human monocyte nucleofactor kit

For RNA interference (RNAi) experiments, primary human monocytes (6 × 10⁶ cells) were transfected with 0.1 nmol of siRNA oligonucleotides (listed in Supplementary Table 1) using a Human Monocyte Nucleofactor Kit (Lonza, VVPA-1007) and the AMAXA Nucleofector System (Lonza) program Y001 for human monocyte transfection according to the manufacturer’s instructions.

Freshly isolated CB-MNCs were transfected with dssiRNA HIF-1α and HIF-2α (Qiagen, Venlo, Netherlands) using ‘Human monocyte nucleofactor kit' (Lonza, VPA-1007). Briefly, for each condition an equal amount of CB-MNCs was centrifuged, and 100 μl nucleofactor was added to the cell pellet. From both dssiRNA HIF-1α and HIF- 2α, 1 μg was added to the cell suspension, and transferred to the cuvette and placed in the electroporation system (Amaxa, Lonza Verviers) according to the manufacturer. Transfected CB-MNCs were resuspended in complete EGM medium, transferred to 0.1% gelatin coated wells, and further cultured under 1% O₂ according the CB-ECFC culture protocol. Mock transfected cells (only electroporation step, no dssiRNA transfection) served as a control in 20 and 1% O₂. Further details are given in Nauta et al. (30 (link)).