Singlet Oxygen

LOCI (marketed by Perkin Elmer as AlphaScreen) is a two-bead assay system: donor beads contain a photosensitizer compound, which upon illumination with laser light at a wavelength of 680 nm converts ambient oxygen to energy-rich, short-lived singlet oxygen; and acceptor beads, which can respond to singlet oxygen with a luminescence/fluorescence cascade leading to an amplified signal in the 520–620 nm range. The oxygen released by a donor bead will only excite acceptor beads within a distance of ~200 nm, and this allows a proximity-based homogenous assay quantifying interacting biomolecules. We have recently described a LOCI-based HTS assay for measuring peptide-MHC class I interaction [13 (link)].
Here, donor beads coated with streptavidin, and acceptor beads for custom protein coupling were both purchased from PerkinElmer. Specific anti-MHC class II monoclonal antibodies were coupled to acceptors beads following the manufacturers recommendation (L243 for HLA-DR; B7/21 for HLA-DP; 9,3F10 for HLA-DQ; and 14-4-4 for I-E^d). Peptide-MHC class II reaction mixtures were generated and incubated 48 h at 18°C as described above for the ELISA. The peptide-MHC class II reaction mixtures were then mixed with equal volumes (12–25 μl) of a solution containing streptavidin donor beads and anti-MHC-II monoclonal antibody conjugated acceptor beads (both at 10 μg/ml in PBS containing 0,1% Pluronic F68). The plates were incubated for 18 h at 18°C and then read in an ENVISION reader (Perkin Elmer). As for the ELISA, the optimal combination of α and β chain concentrations was identified in pilot experiments. Then, peptide titrations and the resulting peptide-MHC class II formations were determined in a LOCI assay calibrated with a known MHC class II standard.
A LOCI-based competitive assay was also developed. In this assay, a binding reaction was set up between a trace concentration of a biotin labeled agonist peptide (biotin attached via a PEG linker to HA_306–318) and non-biotinylated MHC-II molecules. For many HLA-DR molecules one could use low nanomolar (in casu 4 nM) concentrations of agonist peptide. The resulting agonist-MHC-II interactions were developed using a LOCI assay as described above. Once an agonist-MHC-II interaction assay had been established, competition assays using titrations of any test peptide of interest could be conducted.

An ethanolic DMA solution was prepared (0.35 mM) and transferred (190 µL per well) into of a 96-well plate. A DMSO solution (10 µL, 1 mg mL⁻¹) of each extract was added to the respective wells. The absorption of the extract alone (50 µM, ethanol) was measured as well as the suppression of the DMA absorbance in the presence of l-ascorbic acid (5 mM, pH 7.0–7.4). An additional control experiment (extract and l-ascorbic acid) was performed. DMSO (5% in ethanol) was used as solvent control. Berberine (50 µg mL⁻¹, 149 µM) was used as positive control. Excitation was performed with the 96 LED setup, whereby four irradiation steps (468 nm, 5 min each, 6.2 J cm⁻² each) were chosen. The measurement of the absorption was performed with a microplate reader (Tecan, Spark M10) and done in triplicate. The singlet oxygen production after 20 min (24.8 J cm⁻²) was calculated relative to berberine using formula (eqn (2)). A detailed discussion of this formula can be found in the ESI.‡

Using 1,3-diphenylisobenzofuran (DPBF) as the singlet oxygen scavenger and monitoring the changes in absorbance at 410 nm, the singlet oxygen quantum yields of the three PSs were determined by the relative measurement method in MeOH. The calculation formula is as follows:
Φ_△ref is the singlet oxygen quantum yield of reference (MB, Φ_△ = 0.50 in methanol) and K_sam and K_ref are the slopes of change in the maximum absorption of DPBF versus the irradiation time. F is the absorption correction factor, which is expressed by F = 1–10^−OD, where OD is the absorption value at 635 nm.

Intracellular ROS generation induced by NH₂NBS, EtNBS, and NMe₂NBS was detected by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) as an indicator. Singlet oxygen production was confirmed by the singlet oxygen sensor green (SOSG) staining assay. Dihydroethidium (DHE) was used as the indicator to detect the intracellular O₂^−• radical in HepG2 cells. HepG2 cells were incubated with three PSs (1 μM) for 1 h, then the medium was replaced with DCFH-DA (10 μM), SOSG (5 μM), and DHE (5 μM), and incubated for 30 min, respectively. After that, the cells were irradiated with 635 nm laser irradiation for 10 min at a power density of 20 mW/cm². Then the cells were immediately imaged with Zeiss LSM 710. The excitation wavelength for DCFH-DA, SOSG, and DHE was 488 nm, and the emission wavelengths were collected from 500 to 550 nm for DCFH-DA and SOSG, and 570 to 630 nm for DHE.