A Light source Power supply B Arbitrary unit (au) (Relative intensity) Arbitrary unit (au) (Relative intensity) Wave length : : 550-740 nm nm 160 160 120 120 80 80 40 40 5~20 cm cm Fan Fan3737 o C o C Water filter filter Cultured cells cells Lift Lift C 00 500 500 600 600 700 700 800 800 Exposing Wave Length (nm) (nm) TheraBeam VR630: (USHIO LIGHTING, INC.) Light source : : Na-Li lamp Wave length : : 530~630~700 nm nm Power output : : 55 cm: cm: 65mW/cm 2 2 10 10 cm: cm: 43 43 mw/cm 2 2 15 15 cm: cm: 29 29 mw/cm 2 2 18 18 cm: cm: 23 23 mw/cm 2 2 20 20 cm: cm: 20 20 mw/cm 2 2 Power input : : 150 150 W 1~15 24 h 3 h min 1 ~ 24 h FACScan analysis Culture in the presence of FBS Culture with 5-ALA in the absence of FBS Culture in the presence of FBS Light exposure Fig.2
A. Excitation spectra of PpIX B. Emission spectra of PpIX 100 100 50 µm PpIX + Cell 50 µm PpIX + Cell Relative fluorescence intensity Relative fluorescence intensity 0 400 440 480 520 560 600 Excitation Wave Length (nm) 0 400 440 480 520 560 600 Excitation Wave Length (nm) Relative fluorescence intensity 0 430 470 510 550 590 630 670 710 Emission Wave Length (nm) C. Excitation spectra of PpIX D. Emission spectra of PpIX 100 100 U937 (1 mm ALA, 6 h) U937 (1 mm ALA, 6 h) Relative fluorescence intensity 0 430 470 510 550 690 630 670 710 Emission Wave Length (nm)
A Scattergram 0 min 1000 Intact FSC Irradiation time 3 min 5 min 10 min 15 min 0 0 400 800 0 400 800 0 400 800 0 400 800 0 400 800 SSC SSC SSC SSC SSC C Propidium iodide stained Irradiation time 0 min 3 min 5 min 10 min 15 min 200 0Counts Damaged 10 0 10 2 10 4 10 0 10 2 10 4 10 0 10 2 10 4 10 0 10 2 10 4 10 0 FL2-H FL2-H FL2-H FL2-H PI positive 10 2 10 4 FL2-H B Damaged cells (FSC, % of gated) PI positive cells (%) 100 80 60 40 20 0 100 80 60 40 20 0 1 mm ALA 0.25 mm ALA 0 5 10 15 Irradiation time (min) 1 mm ALA 0.25 mm ALA 0 5 10 Irradiation time (min) Fig.7 D 15
A. Scattergram 0 mm B. Annexin and Propidium iodide stained P I (FL-2H) 1000 FSC 0 10 4 UL 10 3 10 2 LL 10 1 0 200 400 600 800 1000 SSC 0 mm UR LR 0.1 mm 0.1 mm 0.25 mm 0 200 400 600 800 1000 0 200 400 600 800 1000 SSC SSC 0.25 mm 0.5 mm 0 200 400 600 800 1000 0 200 400 600 800 1000 SSC 0.5 mm 10 0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Annexin-V-FITC (FL-1H) Damaged cells (%, FSC) Quadrant (%) 100 100 80 60 40 20 80 60 40 20 0 0 0.1 0.25 0.5 ALA (mm) 0 mm 0.1 mm 0.25 mm 0.5 mm UL UR LL LR Fig.8 A. Scattergram 0 min 1000 FSC PI (FL-2H) 0 0 200 400 600 800 1000 SSC 5 min 0 200 400 600 800 1000 SSC 10 min 0 200 400 600 800 1000 B. Annexin and Propidium iodide stained SSC 15 min 0 200 400 60 800 1000 0 SSC 0 min 5 min 10 min 15 min 80 10 4 60 10 3 40 10 2 20 10 1 0 10 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Annexin-V-FITC (FL-1H) Damaged cells (%, FSC) Quadrant (%) 100 80 60 40 20 0 0 5 10 15 20 Irradiation time (min) 0 min 5 min 10 min 15 min UL UR LL LR Fig.9
PI (FL-2H) A. Scattergram 1000 FSC N. I. 0 h 1 h 3 h 6 h 12 h 20 0 0 400 800 0 400 800 0 400 800 0 400 800 0 400 800 0 400 800 SSC SSC SSC SSC SSC SSC 0 0 1 3 6 12 Time after PDT (h) B. Annexin and Propidium iodide stained N. I. 0 h 1 h 3 h 6 h 12 h 10 4 10 3 10 2 10 1 10 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Annexin-V-FITC (FL-1H) Damaged cells (%, FSC) 80 60 40 90 80 70 60 50 40 30 20 10 0 UL UR LL LR Fig.10 Quadrant (%) 0h 1h 3h 6h 12h
1000 FSC A. Scattergram 0 B. TMRE stained 1000 N. I. 0 200 400 600 800 1000 SSC N. I. 3h 0 200 400 600 800 1000 SSC 3h 12h 0 200 400 600 800 1000 SSC 12h Damaged cells (FCS, %) C.Time dependent changes 50 40 30 20 10 0 Damaged cells Membrane potential 0 1 3 12 24 Incubation time (h) 50 40 30 20 10 0 Decrease of membrane potential (FL-2, % of gated) Count 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 TMRE (FL-2H) 10 3 10 4 Fig.11