Video-Rate Bioluminescence Imaging of MMP-2 Figure 7. Identification of MMP2-GLase secretion from the bottom side of cell body. Bioluminescence imaging of HeLa cells transiently expressing MMP2-GLase was performed using an IX81-ZDC Zero Drift microscope system. Objective lens; 606. Luminescence video images were obtained with an exposure time of 500 ms for 50 s after 4 min from the addition of HBSS buffer containing coelenterazine. On recording the luminescence images of MMP2-GLase secretion from cells attached on the cover slip, the in-focus z-axis positions were adjusted to 0, 0.5, 1.0, or 1.5 mm from the upside of the cover slip on a microscope stage using the Zero Drift system. Images of maximum luminescence intensity were made from all frames (100 images for 50 s) at the z-axis positions (Z) of 0, 0.5, 1.0, and 1.5 mm. doi:10.1371/journal.pone.0025243.g007 nal antibody (F-68, Clone No. 42-5D11, Daiichi Fine Chemical Co. Ltd., Toyama, Japan) as follows. HeLa cells (26105 cells) cultured for 24 h in a Falcon 6-well plate (BD Bioscience) were transfected with 2 mg of pcDNA3-hMMP2, pcDNA3-hMMP2- Flag, pcDNA3-hMMP2-GLuc, or pcDNA3 (as a control vector) and 6 ml of Fugene HD (Roche Applied Science), and then cultured for 24 h. To analyze the secreted proteins into conditioned medium, the transfected cells were washed 3 times with HBSS and further cultured in 1 ml of HBSS for 24 h, and then the conditioned medium was 10-fold concentrated using an Ultrafree-0.5 centrifugal filter device with Biomax-10 membrane (MWCO 10 kDa, Millipore, Billerica, MA). The concentrated sample (12 ml) was added with 56 Laemmli’s sample buffer for electrophoresis. To analyze the cell lysate, the cells were washed 3 times with PBS, lysed with 100 ml of 16 Laemmli’s sample buffer, and sonicated for 10 s with an UP50H ultrasonic processor with 1 mm diameter tip (intensity = 0.4, Hielscher, Teltow, Germany). The proteins of the cell lysate and the conditioned medium in 15 ml of the 16 Laemmli’s sample buffer were separated by SDS- PAGE (SuperSep Ace 10% or 5–20% gel, Wako, Osaka, Japan) under reducing conditions. The separated proteins were trans- ferred electrically to a PVDF membrane (Bio-Rad, Hercules, CA) and then the membrane was incubated for 1 h with PBS containing 5% skim milk. After incubation for 1 h at room temperature with the primary anti-GLase or anti-MMP-2 antibodies (1:1000), the membrane was further incubated for 1 h with the secondary HRP-conjugated anti-rabbit or anti-mouse IgG antibodies (1:5000, GE Healthcare UK Ltd). The proteins on the membrane were visualized using an Immobilon Western blotting kit (Millipore) and a LAS-4000mini image analyzer (Fuji film, Tokyo, Japan). Immunofluorescence analysis To determine localization of MMP2-GLase expressed in HeLa cells, immunofluorescence analysis was performed using HeLa cells co-expressed with MMP2-FLAG. HeLa cells (46104 cells) cultured for 24 h in a Falcon 4-well chamber slide (BD Bioscience) was co-transfected with pcDNA3-hMMP2-GLuc and pcDNA3- hMMP2-Flag (0.125 mg each) with 1.5 ml of Fugene HD (Roche Applied Science), and then cultured for 24 h. All procedures for immunostaining were performed at room temperature. The transfected cells were fixed with PBS containing 4% paraformal- dehyde for 20 min, permeabilized for 15 min with PBS containing 0.1% Triton X-100, and then incubated for 30 min with PBS containing 1% fatty acid-free bovine serum albumin (Wako, Osaka, Japan). After incubating for 1 h with the primary anti- GLase rabbit polyclonal antibody (1:1000) and anti-FLAG M2 mouse monoclonal antibody (1:250, F1084, Sigma), the cells were further incubated for 45 min with anti-rabbit IgG antibody conjugated with Alexa Fluor 647 and anti-mouse IgG antibody conjugated with Alexa Fluor 488 (1:2000, Invitrogen), and then mounted in Pro-long Gold antifade reagent (Invitrogen). Fluores- Figure 8. A schematic representation of MMP-2 secretion and binding on the cell surface in migration cells. (A) Hot spots of both secretion and binding of MMP-2 on the cell surface in a migrating cell. (B) Inactive pro-form of MMP-2 is frequently secreted on the leading edge and then bind to micro areas on the cell surface. doi:10.1371/journal.pone.0025243.g008 PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e25243