Nluc substrate

Luciferase assay using the established cell lines was performed as previously reported [35 (link)]. Briefly, XRE-NLuc::HaCaT cells or ARE-NLuc::HaCaT cells (1 × 10⁴) were plated in a 96-well white plate. After the 24-h preculture, treatment with OPAC at the indicated concentrations was carried out for 6 h. Then, 10 μL of the NLuc substrate (#N1120, Promega) was added to the cells. After the 5-min incubation, luminescence was measured using a microplate luminometer (SH-9000Lab, CORONA ELECTRIC, Hitachinaka, Japan). The reporter expression measured as luminescence was corrected based on the number of viable cells obtained as the absorbance.

Relative ATP levels were determined with BTeam, a BRET-based ATP biosensor [20 (link)]. MEF cells were seeded into white 96-well plates at a density of 2000 cells/well and transfected 24 h later using TurboFectin reagent (OriGene, Rockville, MD, USA) with BTeam lacking a targeting sequence (for cyto-ATP determination) or containing a mitochondrial targeting sequence (for mito-ATP determination). After 24 h, cells were treated with H₂O₂ for additional 24 h and then incubated for 30 min in phenol red-free DMEM containing 10% FBS, and 30 µM NanoLuciferase (NLuc) inhibitor to prevent unintended detection of BTeam released from dead cells. Cells were subsequently incubated for 20 min in the presence of NLuc substrate (Promega, Madison, WI, USA) and the luminescence was measured at 520/60 nm (ex/em) (Yellow Fluorescent Protein (YFP) emission) and at 430/70 nm (ex/em) (NLuc emission) at 37 °C. Data are expressed as YFP/NLuc emission ratio.

The dRAPPID assays were done in buffer
(PBS (pH 7.4), 0.1% (w/v) BSA) or in Dulbecco’s modified Eagle’s
medium (DMEM, from Gibco) with phenol red, supplemented with 4.5 g/L d-glucose, 0.58 g/L l-glutamine, 10% fetal bovine serum
(FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (all
from Life Technologies). An assay mixture of 1 nM antibody-LB and
10 nM antibody-SB was incubated with a target analyte for 60 min prior
to the addition of 1000-fold diluted NLuc substrate (Promega, N1110).
For ratiometric detection, 50 or 17 pM of calibrator luciferase was
added to the sensor mixture for IL-6 or TNFα detection, respectively.
The assays were executed in nontreated white Thermo Scientific 384-well
plates (Cat. no 262360) in a total volume of 20 μL. The luminescent
signals were recorded between 398 and 653 nm on a Tecan Spark 10 M
plate reader, with a 25 nm bandwidth, at room temperature. The blue-to-green
ratios were calculated by dividing the blue light emission at 458
by the green light emission at 518 nm. Detection with a digital camera
(Sony DSC-Rx100 III) was done in a gray Styrofoam box. The LOD was
calculated using eq 1, in which SD is the standard error of the y-intercept,
by linear regression of the luminescent signal related to a selection
of low cytokine concentrations.

Cytosolic and mitochondrial ATP levels were quantified as described^{27 (link)}. Cells were transiently transfected with plasmids carrying the bioluminescence energy transfer (BRET)-based ATP biosensor BTeam without targeting signal sequence (for cyto-ATP determination) or targeted to mitochondria (for mito-ATP determination) using TurboFectin reagent (OriGene). The plasmids were a kind gift of Hiroshi Imamura, University of Kyoto. 48 h later, the cells were incubated for 30 min in phenol red-free medium supplemented with 30 μM NanoLuciferase (NLuc) inhibitor to avoid disturbance from the BTeam released from dead cells. Afterwards, NLuc substrate (Promega) was added to the medium and the plate incubated for 20 min. Subsequently, luminescent emissions from the cells were measured at 37 °C at 520/560 nm (Yellow Fluorescent Protein (YFP) emission) and at 430/470 nm (NLuc emission) using a Tecan Spark® Multimode Microplate Reader.

Nluc-ch2C5 was produced by transient transfection with a mixture of pcDNA3.1-ch2C5L and pcDNA3.1-ch2C5H-Nluc. In brief, COS-7 cells in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) were cultured in 24-well plates in a humidified 5% CO₂ incubator at 37 °C. When the cells reached 80% confluence, they were co-transfected with pcDNA3.1-ch2C5L and pcDNA3.1-ch2C5H-Nluc at a ratio of 1:1 (0.25 μg/0.5 μg) using Lipofectamine 3000 transfection reagent (Invitrogen, USA). The cell culture supernatant was collected at 24, 48, 72, and 96 h post-transfection.
To measure luciferase luminescence intensity, 50 μl of cell culture supernatant was mixed with 50 µl of NLuc substrate (Promega, USA) and placed immediately in a GloMax Multi Microplate Reader (Promega, Madison, WI, USA) for luminescence analysis. The Nluc-ch2C5 antibody was purified using protein A/G PLUS agarose (Santa Cruz, CA) and subjected to 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue R250 staining/luciferase luminescence imaging to identify the antibody band.