For chemotaxis assays, migration of explants to Sdf-1 was assessed using a bead assay (Theveneau et al., 2010 (link)). This was done by incubating heparin-acrylic beads (Sigma-Aldrich) overnight at 4°C in PBS supplemented with 1 µg/ml Sdf-1 and placing the beads ∼1 mm apart in a line of silicone grease (VWR) on fibronectin-coated dishes. Explants were then plated perpendicularly at a distance of 250–500 µm. To test the effects of Ca2+ buffering, explants were incubated for 30 min with 50 µM BAPTA-AM (Cambridge Bioscience) or EGTA-AM (AnaSpec). Time-lapse imaging was performed in Danilchick’s medium using an upright microscope (Eclipse 80i; Nikon) fitted with an objective (Plan Fluor 10×/0.30 DIC L/N1) and a camera (ORCA-05G; Hamamatsu Photonics). Data were acquired using SimplePCI software. Tracking of migrating neural crest cells was performed using the ImageJ Manual Tracking plug-in. Immunocytochemistry was performed using a primary rabbit antibody to phosphopaxillin Tyr118 (1:200 dilution; EMD Millipore; Theveneau et al., 2013 (link)). Explants were costained with 2 µg/ml phallodin and 2 µg/ml DAPI.
Orca 05g
Manufactured by Hamamatsu Photonics
The ORCA-05G is a high-performance scientific camera designed for a variety of imaging applications. It features a 5-megapixel CMOS image sensor with a pixel size of 6.5 μm and a maximum frame rate of 100 frames per second. The camera is capable of providing high-quality, low-noise images and offers a wide dynamic range and high quantum efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i, by Nikon (4 mentions)
Plan fluor 10 0.30 dic l n1, by Hamamatsu Photonics (2 mentions)
Simplepci software, by Hamamatsu Photonics (2 mentions)
Heparin acrylic beads, by Merck Group (1 mentions)
Egta am, by AnaSpec (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
Bioscope catalyst afm, by Bruker (1 mentions)
Mzfliii, by Leica (1 mentions)
Dfc420, by Leica (1 mentions)
Im50 v5, by Leica (1 mentions)
Ff01 452 45, by IDEX Corporation (1 mentions)
Ff01 609 181, by IDEX Corporation (1 mentions)
Dg10 1500, by Thorlabs (1 mentions)
Ff510 di02, by IDEX Corporation (1 mentions)
Lab tek chamber, by Thermo Fisher Scientific (1 mentions)
Hcx pl fluotar, by Leica (1 mentions)
L5 filter cube, by Leica (1 mentions)
Dmire2 microscope, by Leica (1 mentions)
Clean microscope coverslips, by Marienfeld (1 mentions)
Ix81 inverted microscope, by Olympus (1 mentions)
8 protocols using orca 05g
1
Chemotaxis Assay for Neural Crest Cells
2
Atomic Force Microscopy of Biological Samples
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Force measurements were made in force-distance mode using a combined BioScope Catalyst AFM (Bruker, Santa Barbara, CA) and an inverted 3-channel Leica TCS SP5 II confocal laser-scanning microscope equipped with an 40 × 0.85NA air objective and Hamamatsu (ORCA-05G) brightfield camera. The AFM was equipped with a temperature controller to keep temperature stable at 37 °C. Triangular tipless gold-coated silicon-nitride cantilevers were used with nominal spring constants of 0.06 N/m as given by the manufacturer (NP-O type D, Bruker). Each cantilever was calibrated before use by the thermal noise calibration method59 (link), 60 (link).
3
Time-lapse Imaging of Cell Behaviors
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For time-lapse recordings, images were captured every 3–5 min for a total of 8 h using Plan Fluor 10×/0.30 DIC L/N1 objectives with DM5500 and DMRXA2 compound microscopes (Leica Biosystems) at 18°C with either a DFC 300FX camera (Leica Biosystems) and LAS acquisition software or an Orca-5G camera (Hamamatsu Photonics) and SimplePCI software. For in vivo imaging, embryos were immobilized onto plasticine. Time-lapse and NCC tracking was performed using the ImageJ Manual Tracking plug-in as previously described (Carmona-Fontaine et al., 2008 (link); Matthews et al., 2008 (link)). Time-lapse imaging for CIL and CoA assays was performed at 18°C in Danilchick’s medium using an upright microscope (Eclipse 80i; Nikon) fitted with an objective (Plan Fluor 10×/0.30 DIC L/N1) and a camera (ORCA-05G; Hamamatsu Photonics). Data were acquired using SimplePCI software. Confocal images were acquired at 22°C in Danilchick’s medium using a TCS SPE upright microscope (Leica Biosystems) fitted with a HC PL APO 20×/0.75 IMM CS2 water objective. ISH images were captured at 18°C using a stereomicroscope (MZ FLIII; Leica Biosystems) fitted with a Plan 1.0×/0.125 objective and a camera (DFC420; Leica Biosystems). Data were acquired using IM50 v5 software (Leica Biosystems).
4
Cell Morphology Imaging in Acute Brain Slices
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For screening cells morphology and checking cell localization in acute brain slices we used a widefield infrared illumination system. This consisted of an IR-LED source (M780L2, Thorlabs) installed at the rear port of a SliceScope Scientifica microscope, an orientable blocking element to create oblique illumination and a condenser focusing the light on the sample. IR light transmitted through the sample was collected with an IR antireflection coated water-immersion objective (Nikon NIR MRD07420 N40X/0.80W) and sent to an IR CCD (IR-1000, DAGE-MIT).
For a first control of ReaChR expression, we performed widefield fluorescence imaging with a system comprising 2 interchangeable LED sources (Thorlabs M470L2, for YFP and M565L3 for dTomato) filtered by 2 interchangeable bandwidth excitation filters (Semrock FF01-452/45 for YFP and F01-545/55-25 for dTomato) and coupled to a diffuser (DG10-1500, Thorlabs) and an achromatic lens (f = 30 mm, #LA1805 Thorlabs). Fluorescence was collected through a tube lens (f = 200 mm), separated from excitation light using a dichroic mirror (Semrock FF510-Di02 for YFP and FF580-FDi01 for dTomato) and detected by a CCD camera (Orca-05G, Hamamatsu) after passing through a visible bandwidth filter (Semrock FF01-609/181 for YFP and FF01-665/150-25 for dTomato).
For a first control of ReaChR expression, we performed widefield fluorescence imaging with a system comprising 2 interchangeable LED sources (Thorlabs M470L2, for YFP and M565L3 for dTomato) filtered by 2 interchangeable bandwidth excitation filters (Semrock FF01-452/45 for YFP and F01-545/55-25 for dTomato) and coupled to a diffuser (DG10-1500, Thorlabs) and an achromatic lens (f = 30 mm, #LA1805 Thorlabs). Fluorescence was collected through a tube lens (f = 200 mm), separated from excitation light using a dichroic mirror (Semrock FF510-Di02 for YFP and FF580-FDi01 for dTomato) and detected by a CCD camera (Orca-05G, Hamamatsu) after passing through a visible bandwidth filter (Semrock FF01-609/181 for YFP and FF01-665/150-25 for dTomato).
5
Time-lapse Imaging of Graft Experiments
6
Time-lapse Imaging of Graft Experiments
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For graft experiments, time-lapse images were acquired every 10-min with an upright microscope (Eclipse 80i; Nikon) fitted with a motorized stage (Prior Scientific) and a camera (ORCA-05G; Hamamatsu Photonics). Images were acquired at 18°C with a 4x objective (CFI Plan Fluor 4x/0.13, WFD= 17.1 mm). Camera, stage, shutter and filter wheels were controlled with a SimplePCI software (Hamamatsu).
7
Live-cell Microscopy of Hoechst-EGFP Samples
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Clean microscope coverslips (Marienfeld) or Labtek chambers (Nunc) containing cells were placed in an incubator equilibrated at 37 °C located on a DMIRE2 Leica Microscope controlled by HCImageLive software (Leica). Images from a 100X (N.A. 1.3–0.6) oil-immersion objective (HCX PL FLUOTAR, Leica) were recorded with a Hamamatsu ORCA-05G camera. A4 (Leica) and L5 filter cubes (Leica) were used for excitation and emission of Hoechst 33342 and EGFP fluorescence, respectively.
8
Mitochondrial Dynamics in Huh-7 Cells
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Huh-7 cells were untreated or treated with 100 nM sangivamycin for 5 days. Cells were then washed with PBS and incubated in serum-free media with fluorescent mitochondrial tracking dyes (2 μM 10 NAO and 0.4 μM MTG) for 30 minutes. Cells were washed with PBS and imaged at 10× original magnification using an IX81 inverted microscope (Olympus Life Science), a fluorescein isothiocyanate (FITC) filter cube, and an ORCA-05G digital camera with a charged-coupled device (CCD) (Hamamatsu Photonics).
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