Modulus microplate multimode reader

Renilla luciferase reporter plasmid DNA (pRL‐TK) and CYP26A1 promoter region cloned firefly luciferase reporter plasmid DNA (pGL4.10) were cotransfected with pcDNA3.1+‐DYK‐tagged RARB or RARB‐mutant plasmids into HEK 293 cells in culture. Genomatix genome analyzer software was used to identify the binding sites for RARB protein in the CYP26A1 promoter region. The cultured cells were lysed in passive lysis buffer and samples prepared as per manufacturer protocol using the Dual‐Luciferase Reporter assay system (Cat # E1960; Promega). Luminescence was measured using a Modulus microplate multimode reader (Model # 9300‐010; Turner Biosystems). The experiments were performed at least six times with three replicates for each sample luminescence reading. Statistical significance was determined by paired t test. Error bars represent standard error of the mean (SEM).

In vitro translation assays were performed as previously described [15 (link)]. 3 pmol of RNA transcripts were used in a 25 μL translation reaction using wheat germ extract (WGE) (Promega) according to the manufacturer′s instructions. The luciferase activity was measured using a luciferase assay reporter system (Promega) and a Modulus Microplate Multimode Reader (Turner BioSystems). At least three independent in vitro translationa ssays were performed for each construct. Standard errors were calculated in Microsoft Excel.

Intracellular survival of Mtb H37Rv was investigated as previously described [17] (link). Briefly, hMDMs were infected with Mtb, containing a pSMT1-plasmid for luciferase-expression, at a ratio of 10 Mtb per hMDM for 1 h at 37°C before hMDM were washed to remove extracellular bacteria, and medium was changed to serum-containing, antibiotic-free DMEM. Following infection hMDMs were stimulated with PMNapo at a ratio of two PMNapo per hMDM. At day 0 (4 hours after addition of PMNapo), 5 and 7 the cell culture supernatants were collected and hMDMs were lysed with water and luciferase expressing Mtb in cell culture supernatants and cell lysates were detected by addition of substrate (1% decanal) before luminescence was measured using a Modulus Microplate Multimode Reader equipped with an injector (Turner Biosystems, Sunnyvale, CA, USA). Data was obtained as arbitrary luminescence units (ALU).

Protoplasts prepared from callus cultures of Arabidopsis thaliana were transfected with luciferase reporter transcripts using a polyethylene glycol-mediated transformation protocol, as previously described [27 (link)]. Briefly, 5 × 10⁶ protoplasts were transfected with 20 μg of OPMV/PEMV2 luciferase reporter transcripts and incubated under constant light for 18 h at 22 °C. The cells were lysed at 18 h post-transfection, and the luciferase activity was assayed with a dual-luciferase reporter assay system (Promega) using a Modulus microplate multimode reader (Turner BioSystems). To check for the stability of the luciferase RNA constructs in protoplasts, the RNA gel blots were conducted using the total RNA extracted from protoplasts at 18 h post-transfection after exhaustive washing was performed.

To collect the peritoneal macrophages, 40 mL of cold, sterile phosphate-buffered saline (PBS, pH 7.2) were injected into the peritoneal cavity for 2 min under massage and immediately collected, as described previously [33 (link),36 (link)]. The cell suspension obtained was centrifuged (speed: 538× g; duration: 10 min; temperature: 4 °C) and resuspended with cold Roswell Park Memorial Institute (RPMI) media without phenol red, supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL streptomycin–penicillin, and 2 mM L-glutamine (all from Sigma-Aldrich, Madrid, Spain). After counting the macrophages using a Spincell hematology analyzer (MonLab Laboratories), 10⁴ cells/well were plated on a black 96-well plate (Thermo Fisher Scientific, Barcelona, Spain) and incubated overnight to allow their attachment to the plate. On the next day, cells were washed with warm RPMI media, and the ROS production was assessed after incubation for 30 min with 20 µM of reduced 2′,7′-dichlorofluorescein diacetate probe (H2DCF-DA; Invitrogen, Paisley, UK) in order to oxidize the H2DCF-DA to a fluorescent compound (2′,7′-dichlorofluorescein) [33 (link),36 (link)]. Then, 0.5 mM of H₂O₂ was added to the plate, and the fluorescence was measured once every 15 min for 2 h by the fluorimeter Modulus Microplate Multimode Reader (Turner BioSystems, Sunnyvale, CA, USA).