HEK293 cells stably expressing hFWE3-EGFP or hFWE4-EGFP were transfected with indicated mCherry constructs and seeded onto collagen-coated glass-bottom dishes for live confocal imaging. For labeling of the plasma membrane, untransfected hFWE3-EGFP or hFWE4-EGFP expressing HEK293 cells were incubated in 1× CellMask Deep Red prior to imaging. Imaging was performed on a Nikon TiE equipped with a Yokagawa Spinning disc using a Plan Apochromat Lambda 100× oil immersion objective lens. During live imaging, sample temperature and CO2 were maintained at 37 °C and 5%, respectively, in a Tokai Hit stage-top incubator.
Hit stage top incubator
Manufactured by Tokai Hit
Sourced in Japan
The Hit stage-top incubator is a laboratory equipment designed to provide a controlled environment for cell culture applications. It maintains stable temperature, humidity, and atmospheric conditions to support the growth and development of cells during microscopic observation or experimentation.
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Lab products found in correlation
6 protocols using hit stage top incubator
1
Live Imaging of FWE-EGFP in HEK293 Cells
2
Real-Time Imaging of Cellular Dynamics
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Cells were grown in an SC medium overnight and diluted 1:20 (vol:vol) the next morning. After 6 hr, cells were immobilized on concanavilin A-coated cover slips and a fresh SC medium was applied. Experimental and control strains were loaded in adjacent imaging chambers, and cells were maintained at 30°C with high humidity using a Tokai Hit stage top incubator. Live cell images were captured using the imaging setup described above with the exception that Z-stacks (8 × 0.3 µm) were acquired every 8 min. We used exposure times of 10 ms (tdTomato) and 200 ms (GFP) for each image. Maximum intensity projections were generated with ImageJ for each timepoint, and figures were created with Nikon Elements software using identical processing steps and settings for each image.
3
Measuring Actin Dynamics in Cardiomyocytes
4
Automated Cell Proliferation Assay
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For cell proliferation assays, cells were seeded at 7,000 cells/well into gelatin-coated 96-well plates and immediately treated with either β-toxin (50 μg mL–1), VEGF (10 ng mL–1), axitinib (10 μM), or mitomycin C (2 μg mL–1). Plates were incubated overnight in a Leica DMi8 equipped with a Tokai Hit stage-top incubator (Tokai Hit Co., Ltd.) set to 37°C, 5% CO2. Merged images were captured every 30 min for 5 h, then every 5 h using a HC PL FLUOTAR 4x/0.13 objective lens. Three independent experiments were conducted for each treatment condition. Cells were automatically counted using ImageJ by modifying the protocol outlined by Venter and Niesler (2019) (link). Images were converted to grayscale and the edges found (Process → Find Edges). An Isodata threshold was then applied (Image → Adjust → Auto Threshold: Isodata) to automatically detect cells. The particle count was then quantified after determining the appropriate particle size to decrease background (Analyze → Analyze Particles [size: 0.003–0.2]).
5
FRAP Analysis of hTR in Cajal Bodies
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Cells were grown on 35 mm glass bottom (#1.5) dishes (MatTek). Prior to each imaging session, cells were transferred to DMEM lacking phenol red and supplemented with HEPES. Imaging was performed on a Nikon Eclipse Ti2 equipped with a Yokogawa Electric CSU-W1 spinning disk module, and using a 100x objective (NA 1.35, Plan Apo). Image acquisition was performed using Nikon Elements software. A Tokai Hit stage-top incubator was used to control temperature, humidity, and CO2 levels. CBs with colocalizing hTR were identified, and a circle of 2 micron diameter around the Cajal body was bleached with 405 nm beam (Bruker). Five initial images were taken of each cell before bleaching. After bleaching, images were taken every 600 ms with a 100 ms exposure time for 60 s. Images were collected using two Andor iXon 88 Life EMCCD cameras connected by a twin-camera module (Cairn). Cells were illuminated with 488 nm light (for MS2-hTR) and 564 nm light (for mCherry-Coilin). Registration was corrected using Tetra Speck beads (Invitrogen) and images were flat-field corrected for the 488 nm channel using a solution of fluorescein (Model and Burkhardt, 2001) . FRAP analysis was performed in FIJI (Schindelin et al., 2012) using the FRAP Profiler plugin (http://worms.zoology.wisc.edu/ research/4d/4d.html). FRAP curves were fit to averages of the datasets in Prism using a one-phase association model.
6
Kidney Development Imaging Protocol
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