Amaxa nucleofection device

BM-MSC nucleofection was performed according to Human MSC Nucleofector Kit (Lonza) protocol. Briefly, 4 × 10⁵ MSC were resuspended in 100 μl of nucleofection buffer with 2 μg pDNA (MC-HLA-G1, pmax-HLA-G1, or pEP-HLA-G1) and pulsed with the U-23 program of the Amaxa Nucleofection device (Lonza). After nucleofection, cells were carefully transferred to MSCGM, and the cell number and percent viability estimated using Trypan Blue. Cells were then replated at 3,000 cells/cm² and kept in culture at 37 °C and 5% CO₂ in a humidified atmosphere. HLA-G1 expression levels were measured by flow cytometric analysis 2 and 10 days after nucleofection. Non-nucleofected cells were used as a control, as were MSC only pulsed with U-23 (no pDNA). Cell recovery and rate of nucleofection were determined using equations previously described by Madeira et al.^{34 (link)} MSC nucleofected with plasmids encoding HLA-G1 were designated nvMSC-HLA-G1.

Cortical neurons were dissociated by papain from postnatal day 2 (P2) Sprague Dawley rats, and co-transfected with a total of 5.0 μg DNA (2.5 μg of each vector used) by electroporation using an Amaxa Nucleofection Device (Lonza). mSOG_1–94 and mSOG-Jα_95–140 were fused to the N-terminus and C-terminus of α-syn in the pCAGGS vector. Co-transfections were done with N-and C-terminus fusions, or C- and C-terminus fusions. The transfected neurons were plated on imaging dishes (P35G-0–14-C, MatTek Corporation) that were coated on the same day with poly-D-lysine. Neurons were cultured in Neurobasal A medium containing 1X B27 Supplements (both from Life Technologies), 2 mM GlutaMAX (Life Technologies), 20 U/mL penicillin, and 50 μg/mL streptomycin for 14 days prior to imaging. Neurons were imaged in HBSS containing 1X B27 Supplements, 25 mM glucose, 1 mM pyruvate, and 20 mM HEPES. All animal procedures were approved by the Institutional Animal Care and Use Committee of UC San Diego. Confocal immunofluorescence images (1024 × 1024 pixels) were acquired on the Olympus Fluoview 1000 laser scanning confocal microscope using a 60X oil immersion objective with numerical aperture 1.42.