D luciferin containing media

Luciferase activity was used to quantitatively measure the in vitro cytotoxic effects of NIR-PIT with huGPR87ab-IR700. For luciferase activity, 200 μL of 150 μg/mL of D-luciferin-containing media (GoldBio, St Louis, MO, USA) was added to PBS-washed cells 24 h after NIR-PIT. The cells were then analysed on a bioluminescence plate reader (Powerscan4; BioTek, Winooski, VT, USA; cat # BT-S4LFPTAD) at 24 h after NIR-PIT, as previously reported [30 (link),35 (link)]

The in vitro cytotoxic effects of NIR-PIT with rova-IR700 were determined via luciferase activity as well as PI staining using flow cytometry. For luciferase activity, 200 μL of 150 μg/mL of D-luciferin-containing media (GoldBio, St Louis, MO, USA) was administered to PBS-washed cells 24 h after NIR-PIT; the cells were then analysed on a bioluminescence plate reader (Powerscan4; BioTek, Winooski, VT, USA). We evaluated luciferase activity in vitro 1, 3, 6, and 24 h after NIR light exposure. With this estimation, we decided to evaluate luciferase activity in vitro at 24 h after NIR-PIT. For the flow cytometric assay, cells were washed with PBS twice and dissociated at 1 h after treatment. The cells were detached from the wells by pipetting, and PI was added to the cell suspension (final concentration, 2 μg/mL) and incubated at room temperature for 30 min before performing flow cytometry. PI fluorescence in 10,000 cells was measured using FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA).