Em ccd c9100 13

ESI-mass spectra for the probes and peptides were measured with an LCMS-2020 system (Shimadzu). HPLC purification and profiling was done with an Inertsil ODS-3 reversed phase column (GL Sciences, 5 μm, 20 × 100 mm and 2.1 × 100 mm), with a linear gradient of 0.1% trifluoroacetic acid in H₂O and 0.1% trifluoroacetic acid in acetonitrile (0−90%) as the solvent, and a flow rate of 3.0 ml/min. All fluorescence spectra were measured with an LS 55 fluorescence spectrometer (Perkin Elmer). Fluorescence spectra were recorded using a FluoroMax^®-4 (HORIBA, Ltd.). The live-cell and fixed-cell imaging were performed using a CellVoyager™ CV1000 Confocal Scanner Box (Yokogawa Electric Corporation) equipped with an ultra-sensitive EMCCD C9100-13 camera (512 × 512 pixels) and an Olympus UPLSAPO 100XO 1.4 oil objective.

Luminescence imaging was performed on an Olympus LuminoView LV200 microscope equipped with an EM-CCD camera (Hamamatsu photonics, EM-CCD C9100-13), a 60-fold oil-immersion magnification objective (Olympus UPlanSApo 60x, NA 1.35, oil immersion) in controlled environment conditions (37 °C, 5% CO₂). Sixteen to 24 h before imaging, 50,000–75,000 cells were seeded on FluoroDishes (WPI, FD35-100) coated with E-cadherin, allowing to obtain a monolayer of individual cells suitable for single cell tracking^{47 (link)}. The medium was supplemented with 0.5 mM luciferin (NanoLight Technology, Cat#306 A) two to four hours before imaging. Fields of view with about 10 to 30 cells were imaged every 5 min with an exposure time of 299 s for 24 to 48 h. To examine propagation of gene expression levels within ES cell colonies (Fig. 6d), 500–1000 cells were seeded on Fluorodishes coated with gelatin, and grown as colonies for 60 h. For each clone, two consecutive images with an exposure time of 5 (Dstn and Jam2) or 3 (pGK) min in at least 10 fields of view were acquired.

Luminescence time-lapse recordings were performed on an Olympus LuminoView LV200 microscope equipped with an EM-CCD cooled camera (Hamamatsu photonics, EM-CCD C9100-13), a 60x magnification objective (Olympus UPlanSApo 60×, NA 1.35, oil immersion) in controlled environment conditions (37 °C, 5% CO₂). To discriminate the luminescence signals from Nluc and Fluc, 700 nm LP filter (Chroma) for Fluc and 460/36 nm band-pass filter (Chroma) for Nluc imaging were used. One day before the experiment, cells were seeded on 35 mm fluorodishes (WPI Inc, FD35–100). Before imaging, the medium was supplemented with 500 μM Luciferin (NanoLight Technology 306 A) and 0.5 µl of RealTime Glo Cell Viability Assay Substrate (Promega G9711). Images were acquired every 3 minutes in the Nluc channel and every 2 minutes in the Fluc channel with a cycle time of 5 minutes up to 24 hours. Cells were recorded for 0–6 hours before stimulation with mouse TGF-β1 (eBioscience, 14-8342-62).
Analyses of intensities from the two channels revealed no detectable bleed-through signal observed in 460/36 nm (Nluc) channel due to luminescence emitted by Luciferin while 1.5% of the Nluc signal was visible in the Fluc channel. This fraction of Nluc signal was subtracted from Fluc signal measured in single cells when analyzing dual-luminescence time-lapse imaging data.