35 mm glass bottom dish

Manufactured by Ibidi

Sourced in Germany, United States

The 35 mm glass bottom dish is a laboratory equipment item designed for cell culture applications. It features a thin glass bottom that allows for high-quality imaging and microscopic analysis of cells grown in the dish.

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Glass bottom dishes (48 citations) µ-Dish 35 mm (37 citations) 35 mm glass-bottom dishes (25 citations) 35-mm dishes (17 citations) Glass-bottom dish (9 citations) 35-mm glass-bottom culture dish (4 citations) μ-Dish 35 mm high Grid-50 Glass Bottom (2 citations)

7 protocols using 35 mm glass bottom dish

Tympanic Membrane Keratinocyte Wound Healing

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Tympanic membrane keratinocytes were seeded into the reservoirs of a two-well culture insert that was adhered to a 35-mm glass-bottom dish (Ibidi, Martinsried, Germany), at a density of 5.0 × 10³ cells/insert, and cultured overnight at 37°C. Once the cells reached confluence, the insert was removed from each dish using a pair of forceps, and the dish was gently rinsed with phosphate-buffered saline (PBS) to remove cell debris. The cells were then cultured in fresh medium, either with or without bacterial supernatant supplementation, and placed into a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Winooski, VT, United States). Phase-contrast images of the wound gaps were viewed and captured under a ×4 objective lens using Gen5 software (BioTek Instruments) at 1-h intervals over 24 h. The wound gap areas at times 0 and 24 h were analyzed with ImageJ software (version 1.46r; National Institutes of Health, Bethesda, MD, United States) to calculate repair rates (expressed in μm²/h).

Zhou K., Tian K.Y., Liu X.Q., Liu W., Zhang X.Y., Liu J.Y, & Sun F. (2021). Characteristic and Otopathogenic Analysis of a Vibrio alginolyticus Strain Responsible for Chronic Otitis Externa in China. Frontiers in Microbiology, 12, 750642.

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Visualization of 6AF-Treated PNT2 Cells

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PNT2 cells were cultured in a 35 mm glass bottom dish (ibidi) until 20% confluency. Cells were treated with 200 μM of 6AF for 72 h at 37 °C in an atmosphere of 5% CO₂. After 3 days, cells were fixed with 1% paraformaldehyde, “click” with a fluorescent tag 7-Azido-4-Methylcoumarin from Sigma-Aldrich with excitation at 387 nm and emission at 470 nm, and membrane stained with Cellmark dye (ThermoFisher, Waltham, MA, USA) with excitation at 522 nm and emission at 535 nm. Images were taken at VetMed Advance Imaging Facility under 100× magnification with oil submersion.

Zhou Q., Xie Y., Lam M, & Lebrilla C.B. (2021). N-Glycomic Analysis of the Cell Shows Specific Effects of Glycosyl Transferase Inhibitors. Cells, 10(9), 2318.

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Imaging Inflammasome-Induced Macrophage Morphology

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To determine cell morphology after inflammasome stimulation, macrophages were seeded in 12-well plates (0.5 × 10⁶/ml per well) and were subjected to treatments indicated. For static image capture, a Nikon Eclipse TS100 microscope was used with 20 × or 40 × magnification. Images were processed with the “NIS-Elements F” software. Representative images were chosen from at least three randomly chosen fields from one experiment out of two independent experiments.
For live cell imaging, DCs seeded into a 35 mm glass bottom dish (Ibidi) were incubated with 5 μg/ml TopFluor-PGPE (Avanti Polar Lipids, inc.) and 200 μg/ml Alexa568-labeled dextran (Thermo Scientific) for one hour. Images were acquired with a Zeiss Axiovert 200M inverted confocal microscope and the level of single cell fluorescent intensity for the two channels was analyzed using ImageJ software (NIH).

Zanoni I., Tan Y., Di Gioia M., Springstead J.R, & Kagan J.C. (2017). By Capturing Inflammatory Lipids Released from Dying Cells, the Receptor CD14 Induces Inflammasome-Dependent Phagocyte Hyperactivation. Immunity, 47(4), 697-709.e3.

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Immunofluorescence of CSF-1 in Bladder Cancer Cells

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BFTC909 and UM-UC-14 cell lines were seeded in a 35 mm Glass Bottom Dish (ibidi) and incubated at 37°C, 5% CO₂. Immunofluorescence was performed using the Image-iT™ Fixation/Permeabilization Kit (Invitrogen™). We removed the culture medium from the cells and then performed cell fixation, permeabilization, and a blocking procedure per the manufacturer's protocol. After blocking, we aspirated the blocking solution and incubated the cells with an anti-CSF-1 monoclonal antibody (M-CSF Antibody (D-4), sc-365779, Santa Cruz) at a 1 : 50 dilution in blocking solution at 4°C overnight. The cells were then incubated with fluorescein isothiocyanate- (FITC-) conjugated secondary antibody diluted in phosphate-buffered saline (PBS) for 1 h at room temperature (protected from light). Next, the cells were incubated with DAPI (Thermo Scientific™) diluted in PBS for 10 min at room temperature in the dark. Finally, cells were mounted by ProLong™ Gold Antifade Mountant (Thermo Scientific™) and observed using a fluorescence microscope.

Hsu W.C., Lee Y.C., Liang P.I., Chang L.L., Huang A.M., Lin H.H., Wu W.J., Li C.C., Li W.M., Jhan J.H, & Ke H.L. (2019). CSF-1 Overexpression Predicts Poor Prognosis in Upper Tract Urothelial Carcinomas. Disease Markers, 2019, 2724948.

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Fluorescence Recovery after Photobleaching

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Fluorescence recovery after photobleaching (FRAP) was performed using Leica confocal TCS SP5 microscope equipped with environment control with LAS-AF software in FRAP mode. H1299 cells were transfected with next plasmids: pEGFP, pΔ133p53βEGFP and pcDNA3mCherryΔNp63α in Ibidi 35 mm glass-bottom dish (ref. 81158). Twenty-four hours post transfection FRAP experiments were performed as shown in the flow chart in Fig. 6a. Imaging was performed in 512 × 512 pixel mode. Pre bleach imaging lasted 5 s. Nuclear bleach was performed applying 5 passes of 1 s using 100% 488 nm laser power on oval ROI (region of interest) with 3 μm diameter in “zoom-in” mode. Thirty post-bleach frames were acquired in bidirectional mode. Twenty cells of each sample per experiment were imagined. Three independent experiments were performed. Recovery curves were normalised and subtracted for background fluorescence. Linear regression of fluorescence recovery curves was calculated and statistical analysis was performed for those with R² higher than 95%.

Arsic N., Slatter T., Gadea G., Villain E., Fournet A., Kazantseva M., Allemand F., Sibille N., Seveno M., de Rossi S., Mehta S., Urbach S., Bourdon J.C., Bernado P., Kajava A.V., Braithwaite A, & Roux P. (2021). Δ133p53β isoform pro-invasive activity is regulated through an aggregation-dependent mechanism in cancer cells. Nature Communications, 12, 5463.

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Quantifying Mitochondrial Superoxide

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To further quantify ROS, we used 5 μM MitoSOX Red (Molecular Probes), which is a specific superoxide marker in mitochondria. To further assay ROS production, cells grown on a 35-mm glass bottom-dish (iBidi) were incubated with 1 μM MitoTracker Green (Molecular Probes) for 15 min at 37°C. Fluorescent intensity and distribution were observed using a Leica DMI6000B.

Na Y.R., Jung D., Song J., Park J.W., Hong J.J, & Seok S.H. (2020). Pyruvate dehydrogenase kinase is a negative regulator of interleukin-10 production in macrophages. Journal of Molecular Cell Biology, 12(7), 543-555.

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Immunostaining of miRNA-transfected MDA-MB-231 cells

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Immunostaining was performed as described previously (Chaluvally-Raghavan et al., 2014 (link); Pradeep et al., 2014 (link)). Briefly, MDA-MB-231 1×10⁴ cells were grown on 35mm glass bottom dish (ibidi Fitchburg, WI, USA) transfected with Cy3 labelled miRNA for 48h then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in blocking solution (0.5% BSA in PBS) followed by blocking for 1 h with 0.5% BSA in PBS, and then stained overnight at 4°C with a primary antibody (1:150 dilution). After washing with PBS, cells were incubated with Alexa Fluor anti-mouse 488 and Alexa Fluor 568 Goat Anti-Rabbit IgG (1:1000, Life Technologies, Carlsbad, CA) for 1 hour at room temperature. Glass slides were mounted using ProLong Gold Antifade Reagent (Life Technologies, Carlsbad, CA) containing DAPI. Each transfection was replicated independently three times. Images were acquired with a 40X objective using a Nikon confocal microscope. Z-stacked sections (10 to 22 slices) of the cells were captured with a motorized Z‐focus controlled camera. ImageJ software was used to reconstruct the images using the Z project plug-in.

Parashar D., Geethadevi A., Aure M.R., Mishra J., George J., Chen C., Mishra M.K., Tahiri A., Zhao W., Nair B., Lu Y., Mangala L.S., Rodriguez-Aguayo C., Lopez-Berestein G., Camara A.K., Liang M., Rader J.S., Ramchandran R., You M., Sood A.K., Kristensen V.N., Mills G.B., Pradeep S, & Chaluvally-Raghavan P. (2019). miRNA551b-3p Activates an Oncostatin Signaling Module for the Progression of Triple-Negative Breast Cancer. Cell reports, 29(13), 4389-4406.e10.

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