Glomax discover plate reader

We validated the resazurin microtitre cell viability assay (REMA) as a screening method, REMA (CellTiter-Blue, Promega, Madison, WI, USA) was used to determine the antimicrobial activities of the drugs.¹¹ (link) Log phase M chimaera cultures were diluted to 1 × 10⁵–5 × 10⁵ CFU/mL, inoculated into a series of drug-free wells containing 2% v/v dimethyl sulfoxide (Sigma Aldrich, St Louis, MO, USA) and into wells containing 2·5 μg/mL rifampicin (50 times the minimum inhibitory concentration [MIC]; Sigma Aldrich, St Louis, MO, USA), and incubated at 37°C for 7 days. A 2% dimethyl sulfoxide concentration was selected to match the final dimethyl sulfoxide concentration of the Pathogen Box screen. To determine antimicrobial activity, CellTiter-Blue was added at a final concentration of 10% v/v and incubated for 16 h. Fluorescence was measured at excitation 580–640 nm and emission 520 nm, using a Glomax Discover plate reader (Promega, Madison, WI, USA). Fluorescence data were corrected for background using media-dimethyl sulfoxide bacteria-free controls. Experiments were repeated a minimum of three times and the datapoints were pooled.

The luciferase assay to quantify YAP activity was performed by transfecting cells with the 8xGTIIC‐luciferase plasmid and using renilla luciferase plasmid (PRL‐TK) as the internal control. Cells transfected only with renilla plasmid were used as a negative control. Briefly, cells were seeded to 80% confluency and transfected with luciferase and renilla plasmid in a 10:1 ratio to assay for YAP activity and prevent over saturation of renilla signal. The Promega Dual‐Luciferase Reporter Assay System (#E1910) was used to quantify luciferase and renilla signal following the manufacturer's protocol. The Promega Glomax Discover plate reader was used to read and record the luciferase signal.

CAR-T cells were sorted by the FACS Aria cell sorting system (FACSCalibur, BD) using GFP. To assess CAR-T cell cytotoxicity, Nalm6-GL and K562-GL cell lines expressing luciferase were plated at 2 × 10⁴ cells/well in black 96-well plates (Corning Costar, Corning, NY, USA) and cultured with T cells/well in an effector to a target ratio of 1:1, 1:2, 1:4, 1:8, and 1:16. Before coculture, T cells were washed three times with phosphate-buffered saline. After 18 h of being cocultured with Nalm6-GL, the medium was aspirated for IL-2, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) secretion analysis using the BD™ Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II (BD Biosciences). The viability of the cancer cells was determined by measuring luminescence with a GloMax Discover Plate Reader (Promega Madison, WI, USA) after adding luciferin (150 μg/ml) to every well.

Gene segments of 1125, 943, 242, 95, and 48 bp located upstream of the major Transcription Start Site (TSS) of exon X1 were obtained by PCR and cloned in Firefly luciferase vector pGL4.15 (Promega, Fitchburg, WI, USA), using Q5 (New England Biolabs) or the high fidelity DNA polymerase Pfu (Stratagene, La Jolla, CA, USA) mixed with Takara Taq. In the same experiments, we used a 187 bp sequence upstream of the TRBV7.2 TSS, as a control promoter. In addition, a 400 bp fragment of the Enhancer ß (Eß) comprising the Eß‐core 7 was cloned in the unique BamH I site of pGL4.15 vectors containing the 943 bp fragment of the X1 promoter or the 187 bp‐long Vß7.2 promoter. The constructs were co‐transfected in triplicates in either HEK293T or Jurkat E6.1 cells with pGL4.75, a plasmid expressing the Renilla luciferase driven by a CMV promoter, as a transfection efficiency control. We used Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and TransIT®Jurkat (Mirus Bio LLC) to transfect HEK293T and Jurkat cells, respectively. Luciferase activities were measured after 24–40 h using the Dual‐Glo® Luciferase Assay System (Promega) and a Glomax Discover plate reader (Promega). The Firefly luciferase activity was normalized to that of the Renilla luciferase and the results compared to those obtained with a promoterless vector, providing the ratios shown in Figure 3A.

Three colonies of Escherichia coli K-12 MG1665 grown in LB agar plates were suspended in 5 mL of LB liquid medium. E. coli suspension was diluted 1:2 (v/v) in sterile PBS or in a pool of cecal content sterile supernatants prepared as described above. E. coli growth in 96-wells plates (100 µL/well) was monitored by measurement of absorbance at 600 nm with GloMax® Discover plate reader (Promega, Madison, WI) every 30 min during 12 h at 37°C under stirring.