Video-Rate Bioluminescence Imaging of MMP-2 Figure 6. Bioluminescence images of MMP2-GLase secretion in a migrating HeLa cell. Luminescence images of MMP2-GLase secretion after the disappearing of luminescence signals of the membrane-associated MMP2-GLase. (A–D) Data of luminescence images after 25 s from the start of recording with 406 objective lens shown in Figure 3 (Video image is in Video S4). (E–G) Data of luminescence images of the leading edge obtained with 1006 objective lens, after the recording with 406 objective lens (Video image is in Video S5). (A) Time-dependent luminescence signals of MMP2-GLase secretion. The first image (T = 0) is one frame in Video S4 at 35 s 54 ms after the start of recording. Luminescence spots newly appeared in a frame are indicated by yellow arrowheads. (B) Magnified luminescence images of the leading edge corresponding to the area indicated by a yellow square in (A). The first image (T = 0) is one frame in Video S4 at 51 s 79 ms after the start of recording. Newly appeared luminescence spots are indicated by yellow arrowheads. (C) An image of the maximum luminescence intensity obtained with an exposure time of 500 ms for 50 s after the disappearance of luminescence signals of MMP2-GLase. The image was made from the frame at 26 s 541 ms to the end frame at 1 min 16 s 118 ms (100 frames) after the start of recording. The luminescence signals of maximum intensity are green-colored. (D) Time-dependent changes of the maximum luminescence intensity in the area of red circles (1–4) in (C). (E) A luminescence image of MMP2-GLase on the leading edge obtained with 1006 objective lens in the area of a yellow square in (C). The image is one frame in Video S5 with an exposure time of 500 ms at 43 s 567 ms after start of recording. The yellow arrows indicate luminescence signals of MMP2-GLase secretion, and luminescence spots newly appeared in this frame of the video image are numbered (spot 1–3). (F) Time-dependent change of magnified luminescence spots in (E). The magnified images of spot 1–3 at 0.5 s correspond to the spots indicated by arrow 1–3, respectively, in (E). (G) An image of the maximum luminescence intensity on the leading edge obtained with an exposure time of 500 ms for 100 s with 1006 objective lens. The luminescence signals of maximum intensity are green- colored and the areas with the strong luminescence spots were red-circled. doi:10.1371/journal.pone.0025243.g006 expression vectors and 6 ml of Fugene HD (Roche Applied Science), and then cultured for 24 h. After washing 3 times with 3 ml of PBS at room temperature, HeLa cells were incubated for 60 min at 37uC with 1 ml of HBSS and 5 ml of the conditioned medium was used for determining the luminescence activity of GLase by addition of 50 ml of HBSS containing coelenterazine (3 mg/ml, Chisso Co., Tokyo, Japan). The initial maximal light intensity was measured using an Atto (Tokyo, Japan) AB2200 luminometer (ver. 2.56, rev. 5.14n) equipped with a R4220P photomultiplier (Hamamatsu Photonics, K.K.). Western blot analysis Western blot analyses were performed using anti-GLase rabbit polyclonal antibody (Prolume) and anti-MMP-2 mouse monoclo- PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e25243