For microscopy analysis, Zeiss LSM 780 confocal scanning light microscopy, with Airyscan technology, was used. This service was provided by the Bio-imaging Facility at VIB Ghent. Microscopy was done using μ-slide 8 well coverslips (ibidi) coated with a 0.01% poly-L-lysine (molecular weight of 70–150 kDa) solution. Cell culture of interest was grown in 5 mL LB medium for 18 h and diluted 10 times in phosphate-buffered saline (PBS). Subsequently, 500 μL of this medium was dyed with 1 μL of the red FM4-64 membrane dye (1 mg/mL stock) (Thermo Fisher Scientific). The dyed cell culture was applied on the μ-slide 8 well coverslip and incubated for 30 min at room temperature. Next, the coverslip well was pipetted until dry and 2–3 droplets of mounting medium (1% n-propyl-gallate in glycerol) were added. The image processing package Fiji v1.8.0172, a distribution of ImageJ, was used to compose intensity profiles.
μ slide 8 well coverslips
Manufactured by Ibidi
The μ-slide 8 well coverslips are glass slides with eight individual wells, designed for cell culture applications. Each well provides a clear, flat surface for cell growth and observation under a microscope.
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Lab products found in correlation
4 protocols using μ slide 8 well coverslips
1
Confocal Microscopy Protocol for Cell Imaging
2
Visualizing MED25 in HeLa Cells
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HeLa cells were seeded on μ-slide 8-well coverslips (Ibidi) and transfected with NS1-GFP (100 ng per well) using FuGENE in accordance with the manufacturer’s instructions. The next day, the cell medium was removed and the cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The cells were blocked and immunostained with an antibody specific for MED25 (Atlas Antibodies; HPA068802) and Alexa Fluor-568 conjugated secondary antibody. Nuclei staining was performed using DAPI. Images were acquired using a Zeiss LSM 880 Fast Airyscan.
3
Visualizing RSV Protein Interactions
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A549 cells were seeded on μ-slide 8-well coverslips (Ibidi) and infected with RSV mKate2-BirA*-Flag or RSV NS1-BirA*-Flag at an MOI of 1 or were mock infected for 4 h at 37°C. After 4 h, the virus inoculum-containing medium was replaced with DMEM supplemented with 2% FBS and 50 μM biotin for an additional 20 h. The cell medium was subsequently removed, and the cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The cells were blocked and stained with streptavidin-AF488. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using a Zeiss LSM 880 Fast Airyscan.
4
Cell Migration Dynamics Quantification
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Cells were seeded onto μ-slide 8 well coverslips (Cat. #: 80821, ibidi), at a density of 1500 cells/cm2, approximately 16–24 h prior to imaging. Coverslips were coated with human plasma fibronectin diluted to a concentration of 5 μg/cm2 with 1× PBS for 1 h at 37 °C. Images were acquired in phase contrast every 10 min for 24 h on a Zeiss AxioObserver fully automated inverted microscope equipped with a Plan-Neofluar 10x/0.3NA Ph1 objective, Axiocam 506 camera (Carl Zeiss, Jena, Germany), and Chamlide TC-L-Z003 top-stage incubator system (Live Cell Instrument, Seoul, South Korea). Cells were then manually tracked using MetaXpress analysis software (v. 5.3.0.5; Molecular Devices, Sunnyvale, CA). X, Y position data for each cell track was exported to MATLAB (v. 8.6.0, rel 2015b; The MathWorks, Natick, MA) for further analysis. Rose plots depicting cell movement were created by superimposing the starting position of each cell track on the origin (0, 0). The average speed of each cell was calculated by determining the mean distance traveled between each time point over the imaging interval (10 min). A histogram of cell speed was created in Prism 7 (v. 7.0a; GraphPad Software, La Jolla, CA) by binning data into 5 μm/h intervals ranging from 10 to 70 μm/h. The data was subsequently smoothed using a spline curve and plotted against relative frequency.
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