Live cell imaging was performed in an environmentally controlled FCS2 live cell chamber (37°C temp and 5% CO2) (Bioptechs, Butler, PA) system using a confocal microscope (Nikon A1, Nikon) controlled by NIS-Elements software. Prior to imaging, 2×105 cells were plated on 35 mm glass bottom microwell dish (Mat-Tek, Ashland, MA) and incubated for 24 hours. Cells were then incubated for 2-3 minutes with Hoechst 33342 in complete media to label nuclei. The dishes were then immediately assembled into a heated chamber and time-lapse imaging was initiated immediately. The Z stacks images of selected cells were acquired every 5 minutes using excitation at 488 nm (for GFP) and 403.8 nm (for Hoechst 33342). Fluorescence was captured through an Plan Apo VC 60× oil immersion objective, NA = 1.40 (Nikon) by using perfect focus system to correct possible focus drift during time lapse imaging. Culture medium containing BITC was used, and the temperature of the chamber was maintained at 37°C during the imaging. GFP excitation was kept as low as possible to avoid photo-destruction of the cell. For all studies, 3-6 fields per dish and duplicate dishes per condition were evaluated in three independent experiments. Images were processed using MetaMorph (Molecular Devices, Sunnyvale, CA), and Adobe Photoshop.
35 mm glass bottom microwell dish
Manufactured by MatTek
Sourced in United States
The 35 mm glass bottom microwell dish is a laboratory equipment designed to provide a transparent surface for cell culture and microscopy applications. It features a glass bottom that allows for high-quality imaging and observation of cells under a microscope.
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Lab products found in correlation
Plan apo vc 60, by Nikon (1 mentions)
Fcs2 live cell chamber, by Bioptechs (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Ab150113, by Abcam (1 mentions)
Plan apochromat 63 1.4 oil objective, by Zeiss (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Sp8x confocal microscope, by Leica (1 mentions)
6 protocols using 35 mm glass bottom microwell dish
1
Live Cell Imaging of GFP Dynamics
2
Visualizing uPAR Expression and CAR-T Binding
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Cell lines (HEK-293T-uPAR-T2A-Luc and SNU-216) cultured on a 35-mm glass-bottom microwell dish (MatTek Corporation) were fixed with 4% paraformaldehyde for 10 min. Both the cell lines and antigen-recovered paraffin section of xenografts derived from the DGC patient A were subjected to permeabilization with 0.1% Triton X-100, followed by incubation with primary antibodies, the anti-uPAR mAb, and anti-human CD8a (1:200; Sigma-Aldrich, ZRB1010), overnight at 4°C. Then, the samples were washed thrice with PBS and incubated with fluorescent-labeled secondary antibody (Abcam, ab150113). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole before mounting. Confocal fluorescence images were captured using a Zeiss LSM880 laser microscope (Plan-Apochromat 63× /1.4 oil objective).
To determine the ability of uPAR CAR-T cells to bind to HEK-293T-uPAR-T2A-mCherry cells, they were cocultured on a 35-mm glass-bottom microwell dish for 4 hours. Confocal fluorescent images were captured using a Zeiss LSM880 laser microscope.
To determine the ability of uPAR CAR-T cells to bind to HEK-293T-uPAR-T2A-mCherry cells, they were cocultured on a 35-mm glass-bottom microwell dish for 4 hours. Confocal fluorescent images were captured using a Zeiss LSM880 laser microscope.
3
Fluorescent Labeling of P. tricornutum
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For confocal microscopy, fluorescent labeling were performed on living or fixed P. tricornutum cells. After the different steps of labeling and rinsing, 5 μL of the diatom cell solution were deposited on a 35-mm glass bottom microwell dish (MatTek corporation) and covered with a small agar pad (Fisher, 0.3 g/20 mL) to stabilize microalgae during imaging. Acquisitions were performed at room temperature with an inverted Leica TCS SP5 confocal microscope (Leica Microsystems, Nanterre, France).
4
Slice Culture and EdU Incorporation
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Slices were prepared as previously described 6 , 61 . The slices were immersed in collagen and transferred with ~ 200 μl of collagen solution into the 14‐mm well of a 35‐mm Glass Bottom Microwell Dish (MatTek). The dish was placed for 5 min on a heating block at 37°C. This transfer was defined as t = 0 of slice culture and was followed by 40 min in the slice culture incubator to allow the collagen to solidify. The dish then received 2 ml of SCM, and slice culture was continued in the slice culture incubator for the indicated times (24 or 48 h). For the EdU incorporation experiments, EdU (final concentration 2.5 μg/ml) was added to both collagen and SCM.
5
Subcellular Localization of Labeled Defensins
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MtDef5 and MtDef5B were each labeled with DyLight550 amine-reactive dye according to the manufacturer’s instructions (Thermo Scientific, United States). The assay was performed in 10 mm microwell of 35 mm glass bottom microwell dish (MatTek Corporation, Ashland, MA, United States). The X. campestris bacterial cells (final test concentration of 1 × 107 cfu/ml) were either treated with DyLight550-MtDef5 or DyLight550-MtDef5B (1 × MIC) and co-stained with nucleic acid selective dye SYTOX Green (SG) (1 μM) and incubated in dark at 28 ± 2°C on a rotary shaker at 125 rpm for different time points (5–60 min). Internalization and subcellular localization of each DyLight550-labeled defensin were visualized using the Leica SP8-X confocal microscope (100× magnification). The DyLight550-labeled defensin was excited at 550 nm and fluorescence was detected at 560–600 nm, whereas SG was excited at 488 nm and fluorescence was detected at 510–530 nm.
6
Measuring Mitochondrial Membrane Potential
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