Multi sample plate reading luminometer

HEK293T cells were seeded in 6-well plates (1×10⁵ cells/well) one day before transduction with 500 ng p24 IDLV-ZFN(AAVS1)/fluc or IDLV/fluc. Luminescence analysis was carried out 3, 10 and 18 days after transduction, respectively. 0.5×10⁵ cells were transferred to 96-well plates 6 hr before luciferase activity analysis using ONE-Glo luciferase assay system (Promega, Madison, WI). Luminescence analysis was performed on a multi-sample plate-reading luminometer (Berthold Technologies, Bad Wildbad, Germany).

Dual-Glo luciferase assays were performed as previously described in Bak et al.¹¹ (link) and Hollensen et al.¹⁵ (link). In brief, HEK293 or HeLa cells were seeded in white 96-well plates at a density of 3,000 cells/well or 5,000 cells/well, respectively, 1 day before transfection. All cotransfections were carried out using X-tremeGENE 9 (Roche, Basel, Switzerland) according to manufacturer’s protocol, 6 ng of psiCHECK reporter plasmid, and 102 ng miRNA inhibitor plasmid. However, for experiments shown in Figures 1A, 1B, 4B, S3A, and S3B, 102 ng ciRS-7 expression plasmid and equal molar amounts of the other used miRNA inhibitor plasmids were used. Due to relative low endogenous expression of miR-7, miR-143, miR-145, and miR-203,11 (link), 14 (link) 3 ng of miRNA expression plasmid were included for studies of suppression of these miRNAs. Two days after transfection, luciferase expression levels were measured using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA) according to manufacturer’s protocol on a multisample platereading luminometer (Berthold, Bad Wildbad, Germany). Data are presented as RLuc expression levels relative to the FLuc expression levels and normalized to a negative control.