Synergy two microplate reader

Huh-7.5 cells were seeded on 96-well clusters and transfected with in vitro transcribed subgenomic HAV-FLuc replicon or a replication-incompetent mutant (González-López et al., 2018 (link); Yi and Lemon, 2002 (link)) using TransIT-mRNA transfection kit (Mirus Bio, #MIR2250) according to the manufacturer’s instructions. Cells were harvested in 1 × passive lysis buffer (PLB, Promega) at the indicated times post-transfection and luciferase activity was measured using a firefly luciferase assay system (Promega, #E1501). For HAV-NanoLuc assays, cells were lysed for 5–10 min in 1 × PLB and mixed with 1 × substrate for Oplophorus luciferase (NanoLight Technology, #325) according to the manufacturer’s instructions. All luciferase readings were obtained on a BioTek Synergy two microplate reader.

To understand the population structure of S. pneumoniae causing ocular infections, isolates included in this study were submitted to whole-genome sequencing. Total DNA was purified from an overnight pure culture in 5 ml Todd Hewitt broth using the DNeasy DNA extraction kit (Qiagen, Valencia, CA). DNA quality was verified on a Bio-Tek Synergy two microplate reader (Winooski, VT) prior to quantification using a Qubit fluorometer and dsDNA High-Sensitivity assay kit (Invitrogen, Carlsbad, CA). Library preparation for Illumina sequencing was carried out using the Nextera XT DNA Library Preparation kit (Illumina, San Diego, CA), according to the manufacturer’s specifications. Quality and quantity of each sample library was measured on a TapeStation instrument (Agilent Technologies, Santa Clara, CA). The genomes were sequenced as 2×100 bp or 250 bp reads on an Illumina HiSeq sequencer, according to the manufacture’s specifications with a minimum depth of coverage of 30× (ranging from 30× to 855×; median 243×). Sequence reads were assembled de novo using CLC Genomics Workbench (CLC Bio, Cambridge, MA). Samples with sequence reads below a quality score of 25 at any position and assemblies that were not between 1.8 to 2.4 Mb were re-sequenced. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the BioProject number PRJNA715222.

Cultured MMVECs from either ecARNT-/- or control mice were seeded at a density of 2.0 × 10⁴ cells in 96 well plates. Following incubation with NG or HG media for 48 h, the ROS levels were assessed using the Cellular ROS/Superoxide Detection Assay Kit (ab139476 from Abcam), which has been widely and successfully utilized in many different studies (Hu et al., 2019 (link), 2020 (link); Ren et al., 2019 (link); You et al., 2020 (link)). The cells were prepared for measurement as instructed by the manufacturer’s manual and submitted to the Biotek Synergy two microplate reader set with standard fluorescein (Ex = 488 nm, Em = 520 nm) and rhodamine (Ex = 550 nm, Em = 610 nm).