Microscope stage top incubation chamber

Manufactured by Tokai Hit

Sourced in Japan

The microscope stage-top incubation chamber is a device designed to maintain a controlled environment for live-cell imaging and analysis under a microscope. It maintains stable temperature, humidity, and gas levels to support cell viability during extended observation periods.

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Lab products found in correlation

Electron multiplying ccd camera, by Oxford Instruments (1 mentions) Nipkow disk confocal scanner unit csux1, by Yokogawa (1 mentions) Celltracker green, by Thermo Fisher Scientific (1 mentions) Te2000 e2 inverted microscope, by Nikon (1 mentions) Gfr matrigel, by Corning (1 mentions) Microscope cover glass, by Thermo Fisher Scientific (1 mentions) Rhodamine 110, by Merck Group (1 mentions) Rhodamine b, by Merck Group (1 mentions) Six well plate, by Corning (1 mentions)

3 protocols using microscope stage top incubation chamber

Live-Cell Imaging of Rab-Mediated Trafficking

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Madin-Darby canine kidney cells were seeded in ϕ3.5 cm glass bottom dishes and were co-transfected with HA-EGFP and NA-mSB expression plasmids. MDCK cells stably expressing AcGFP-Rab were similarly cultured in ϕ3.5 cm glass bottom dishes and were transfected with the NA-mSB expression plasmid. Before live-cell imaging, the culture medium was replaced with Dulbecco’s modified Eagle’s medium without phenol red (Life Technologies) supplemented with 10% FBS and the cells were placed in a microscope stage top incubation chamber (Tokai HIT, Japan). Live-cell imaging was performed at 12 hpt using a confocal microscope (IX71, Olympus Optical, Japan) equipped with an oil immersion objective (Plan Apo N, 60×, 1.42NA, Olympus Optical) and a microlens-enhanced Nipkow-disk confocal scanner unit (CSUX1, Yokogawa Electric, Japan). Sequential images with excitation at 488 and 568 nm were acquired at 1-s intervals for 100 s (100 exposures each for GFP and RFP) by an electron multiplying CCD camera (Luca, Andor Technology, United Kingdom). Bleach and contrast corrections of acquired images were performed using ImageJ software, and tracking of punctate fluorescent signals using MTrackJ plugin created by Eric Meijering¹.

Sato R., Okura T., Kawahara M., Takizawa N., Momose F, & Morikawa Y. (2019). Apical Trafficking Pathways of Influenza A Virus HA and NA via Rab17- and Rab23-Positive Compartments. Frontiers in Microbiology, 10, 1857.

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Live Imaging of Pericyte-Endothelial Cell Interactions

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Sorted pericytes (CD146⁺/CD90⁺/CD56⁻/CD45⁻/CD34⁻/CD31⁻) (passage <8) were maintained in basal growth medium and kept at <70% confluence. Cells were then seeded at a density of 1.2 × 10⁴ cells/cm² on growth factor reduced (GFR)-Matrigel (Corning)-coated 24-well plates in endothelial growth medium on GFR-Matrigel. Pericytes were also separately seeded on Matrigel substrates in co-culture in a 1:10 ratio with primary human pulmonary artery endothelial cells (hPAECs, P8) (Lonza). For co-culture experiments, populations were labeled with Cell Tracker Green (pericytes) or Red (hPAECs) (Invitrogen). Cells were cultured at 37°C and 5% CO₂ in a humidified microscope stage-top incubation chamber (Tokai Hit). Cells on Matrigel were visualized using phase-contrast and epifluorescence microscopy on a Nikon TE-2000-E2 inverted microscope equipped with FITC and tetramethylrhodamine isothiocyanate filter cubes and a 10× objective, and images were captured every 10 min for up to 50 hr using a CoolSNAP ES2 Monochrome 1,394 × 1,040 High Resolution Camera and NIS Elements Software 3.2. Images were also captured after 1, 3, and 5 days of culture.

Billaud M., Donnenberg V.S., Ellis B.W., Meyer E.M., Donnenberg A.D., Hill J.C., Richards T.D., Gleason T.G, & Phillippi J.A. (2017). Classification and Functional Characterization of Vasa Vasorum-Associated Perivascular Progenitor Cells in Human Aorta. Stem Cell Reports, 9(1), 292-303.

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Imaging Comet Incubation Conditions

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In order to create an environment similar in geometry to the comet incubation conditions, but with a bottom surface more amenable to fluorescence and thermal imaging, one 35mm-diameter well was cut from a six-well plate (Corning, Corning, NY) using a razor blade, and the polystyrene bottom was replaced with a microscope cover glass (Fisher, Waltham, MA), attached around the outside edge of the well with silicone adhesive (DAP, Baltimore, MD). The dye solution was composed of 0.1mM rhodamine B (Sigma) and 0.1mM rhodamine 110 (Sigma) in PBS-BSA, for a pH-buffered solution comparable to the infection media from the comet assay in terms of ionic composition, viscosity, and surface tension. Two mL of dye solution was placed in the glass-bottom well, a loosely-fitting cover was placed on the dish, and the covered dish was placed inside a microscope stage-top incubation chamber (Tokai Hit, Shizuoka-ken, Japan). The incubation chamber was placed on the microscope stage with temperature and CO2 at ambient conditions. The dish was incubated 3.5 hours to allow the system to approach a thermal and flow steady-state condition.

Lindsay S.M, & Yin J. (2016). Temperature gradients drive radial fluid flow in Petri dishes and multiwell plates. AIChE journal. American Institute of Chemical Engineers, 62(6), 2227-2233.

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