Neon-IFT88 or Neon-Kif3B-stable NIH3T3 cells were transfected with the indicated constructs and then seeded onto poly(d -lysine)-coated borosilicate glass Lab-Tek eight-well chambers (Thermo Scientific). The cells were imaged every 200 ms for 30 s on a Nikon T1 inverted fluorescence microscope (Nikon) with a ×60 oil objective (Nikon), DS-Qi2 CMOS camera (Nikon), and 37 °C, 5% CO2 heat stage (Live Cell Instrument). Time-lapse images were processed by Huygens deconvolution (Scientific Volume Imaging), and kymographs were produced with ImageJ and the plug-in KymographClear70 (link).
Lab tek eight well chambers
Manufactured by Thermo Fisher Scientific
The Lab-Tek eight-well chambers are a versatile laboratory equipment designed for cell culture applications. These chambers provide a controlled environment for culturing cells and performing various experiments. Each chamber features eight individual wells, allowing for the concurrent testing of multiple samples or conditions. The chambers are constructed with high-quality materials to ensure reliable performance and consistent results.
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Lab products found in correlation
Ds qi2 cmos camera, by Nikon (2 mentions)
T1 inverted fluorescence microscope, by Nikon (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Element ar software, by Nikon (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Poly dlysine coated, by Thermo Fisher Scientific (1 mentions)
4 protocols using lab tek eight well chambers
1
Live-cell imaging of cytoskeletal dynamics
2
Isolation and Culture of Zebrafish Cardiomyocytes
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Cardiomyocytes from dissected ventricles of bag3e2/e2 mutants and WT control fish were dissociated as reported (Warren et al., 2001 (link)). Dissociated cardiomyocytes were then resuspended in L-15 medium containing 10% fetal bovine serum (Invitrogen) and placed in Lab-Tek eight-well chambers (Thermo Fisher Scientific). Healthy dissociated cardiomyocytes were usually attached to the chamber within 1 h, and the attached cardiomyocytes were then cultured at 28.5°C for 12 h, followed by α-actinin antibody immunostaining to confirm their cardiomyocyte identity (Yang and Xu, 2012a (link)). Images of α-actinin-stained cardiomyocytes were captured, and the cardiomyocyte area was measured by outlining each individual cardiomyocyte using ImageJ software.
3
Live-cell Fluorescence Microscopy Protocol
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Cells were plated on poly(d -lysine)-coated borosilicate glass Lab-Tek eight-well chambers (Thermo Scientific). Live-cell imaging was performed using a Nikon T1 inverted fluorescence microscope (Nikon) with a ×60 oil objective (Nikon), DS-Qi2 CMOS camera (Nikon), and 37 °C, 5% CO2 heat stage (Live Cell Instrument). Imaging was acquired using Nikon element AR software. Images with multiple z-stacks were processed with Huygens deconvolution (Scientific Volume Imaging), and the maximum intensity projections of images were produced by Nikon element AR software (Nikon). The image analysis was mainly conducted by Nikon element AR software (Nikon).
4
Fluorescent Labeling of Transfected HEK293T Cells
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HEK293T cells transfected with Venus-mPrestin(N7T, N308S) were seeded on poly-Dlysine-coated Lab-Tek eight-well chambers (Thermo Scientific). Transfected cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 15 min and subsequently permeabilized by 0.1% Triton X-100 (Sigma-Aldrich). After incubation of blocking solution (PBS with 2% bovine serum albumin) for 30 min at room temperature, cells were stained with phalloidin Alexa Fluor 594 (1:100 dilution; Thermo Scientific, A12381) or anti-𝝰-tubulin antibody (1:1000 dilution; Sigma-Aldrich, T6199) for 1 h at room temperature.
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Goat anti-mouse IgG Alexa Fluor 594 (1:1000 dilution; Thermo Scientific, R37121) were incubated with cells for 1 h at room temperature.
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Goat anti-mouse IgG Alexa Fluor 594 (1:1000 dilution; Thermo Scientific, R37121) were incubated with cells for 1 h at room temperature.
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