Amaxa nucleofactor kit

FHC silencing of K562 cells was performed using two different methods: (i) stable transduction of a lentiviral DNA containing an shRNA targeting the 196–210 region of the FHC mRNA (sh29432) (K562^shFHC) or a control shRNA without significant homology to known human mRNAs (K562^shScr) and (ii) transient transfection of a pre-cast siRNAspecific for FHC (K562^siFHC) or a negative control siRNA (K562^cntr) (Thermo Fisher Scientific). Stable transduction was achieved, as previously reported [19 (link),23 (link)]. Transient transfections were performed using the Amaxa Nucleofactor kit (Lonza, Basel, Switzerland) [37 (link)]. FHC stable knock-down was verified by Western Blot analysis and RT-qPCR while FHC transient knockdown was checked at 24, 48 and 72 h by RT-qPCR analysis. FHC-reconstitution in K562^shFHC cells was performed using the expression vector containing the full length of human FHC cDNA (pc₃FHC) (K562^shFHCpc₃^FHC).
K562^shFHC cells were also transiently transfected with a final concentration of 100 nM of a specific miR-150 inhibitor (K562^{shFHCmiR−150 inhibitor}) obtained from Thermo Fisher Scientific, using the Amaxa Nucleofactor kit (Lonza). 48 h after transfection, miR-150 levels were measured by TaqMan microRNA Assay (Thermo Fisher Scientific).

Microfluidic devices were generated as described previously6 (link). Embryonic rat cortical neurons (E17) were electroporated with BDNF-mCherry using Amaxa Nucleofactor kit (Lonza). Neurons were then plated in the proximal chamber in Neurobasal supplemented with 2% B27, 1% GlutaMAX and 1% penicillin/streptomycin. Videomicroscopy and immunolabelling acquisitions were done in the distal part of the microgrooves 3–5 DIV, when axons have already crossed the microchannels (Fig. 2c).