Glomax discover multimode

10 mg of frozen homogenate brain tissue was resuspended in 100 μL of PBS and processed using the Amplex red cholesterol assay kit (Life Technology, Monza, Italy), according to the manufacturer’s instructions. Absorbance was measured by using the GloMax^® Discover multimode plate reader (Promega, Italy) at 490 nm. Cholesterol concentrations were evaluated by using a standard curve, according to the manufacturer’s instructions.

Autologous anti-GD2 CAR T or GFP T cells were used as effectors (E) and human GD2-positive primary cell lines as targets (T). Different effector-to-target (E:T) ratios were used to assess the cell viability using 96-well black plates (Corning, Kennebuck, ME, USA; #3904) and GloMax Discover Multimode as microplate reader (Promega, Madison, WI, USA). Cultures containing the medium alone or 1% Triton X-100 were used as controls, representing 100% and 0% cell viability, respectively. Average viability was calculated as 100 × (experimental fluorescence − 0% viability fluorescence)/(100% viability fluorescence − 0% viability fluorescence). Co-cultures were also monitored by EVOS FL auto (Thermo Fisher Scientific) over 72 h.

Promoter fragments (1500 bp) upstream of the FBN2 or VPS39 transcription start sites (TSS) were cloned into a CpG-free luciferase reporter vector (pCpGL-basic)^{72 (link)} by GenScript (GenScript USA Inc., Piscataway, NJ, USA). The resultant plasmids were in vitro methylated using the methyltransferase M.SssI (New England Biolabs, Frankfurt, Germany) (2.5 U/μg DNA), or mock-methylated (Control).
C2C12 myoblasts were cultured in 96-well plates in DMEM medium with 4.5 g/L glucose supplemented with 10% horse serum, and co-transfected with 50 ng (FBN2) or 150 ng (VPS39) methylated plasmid DNA or mock-methylated plasmids together with 4 ng Renilla luciferase control reporter vector (pRL-CMV vector; Promega, Madison, WI, USA) using FuGene HD (Promega). Luciferase activity was measured 48 h later using dual-luciferase reporter assay (E1910, Promega) and a GloMax Discover Multimode microplate reader (Promega) according to the manufacturer’s instructions. Briefly, the cells were lysed in 100 µl PBL-buffer, agitated on an orbital shaker for 25 min (600 rpm). Luminescence was measured in 3.5 µl of lysate using 65 µl each of Assay Reagent II, and Stop and Glo reagent. An average firefly/Renilla signal was calculated from triplicates for each experiment and condition (three technical replicates for each experiment, and each experiment is done with n = 5–6).