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. 2019 Feb 14;9:2084. doi: 10.1038/s41598-019-38575-x

Figure 4.

Figure 4

Combination of pan IR700 and tra IR700 mediated PIT is more cytotoxic than either agent alone in vitro. (A) Determination of LD50 of NIR light for combination PIT. 15 μg/mL (100 nM) of antibody was added to 1 × 105cells/mL and incubated overnight. Cells were then irradiated with NIR light at 0 J/cm2, 4 J/cm2, 10 J/cm2, 20 J/cm2, 40 J/cm2, 64 J/cm2, 80 J/cm2, or 100 J/cm2 the following day. Cell survival was then measured 24 h later using Promega CellTiter-Glo Luminescent Cell Viability Assay. The LD50 for the combination PIT was determined to be 28.66 J/cm2 compared to 71.55 J/cm2 for pan IR700 with NIR arm, demonstrating potent phototoxicity in the combination group. LD50 for tra IR700 with NIR could not be determined due to insufficient cell death over 0–100 J/cm2 range of NIR irradiation. IR700 (200 nM) showed no overt toxicity. (B) Conjugated antibodies do not show any effects without NIR. IR700 conjugated or unconjugated panitumumab, trastuzumab, or panitumumab + trastuzumab are not cytotoxic at (i) 24 h or (ii) 72 h in vitro. 5 × 103 SW780 cells/well were seeded overnight in a 96-well plate. Cells were then incubated with 100 nM antibody solution or 200 nM (100 nM pan or pan IR700 and 100 nM tra or tra IR700) in the combination well or 200 nM of IR700 Dye700Dx for 24 h or 72 h. Cell survival was measured using Promega CellTiter-Glo Luminescent Cell Viability Assay. No antibody or IR700 mediated cytotoxicity was observed between the groups at 24 h and 72 h.