Glass bottom μ slide, by Ibidi - Product details

1

Raman Spectroscopy of C. pneumoniae Infected Monocytes

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Monocytes infected with C. pneumoniae (2 × 10⁴ IFU) were cultured for 6 and 48 h on glass bottom μ-slides (170 μm thickness; ibidi GmbH, Munich, Germany). Samples were fixed with 4% paraformaldehyde for 4 min and washed 3 times with PBS. Raman spectroscopy was performed by CellTool (Bernried, Germany) using the Bio-Ram® system and the Bio-Ram® software. Raman spectra of 60 cells per assay were recorded with a 785 nm laser (80 mW), applying an accumulated time of 3 × 10 sec. Data of the biologically relevant region (700–3000 cm⁻¹) were pre-treated with a median filter for noise reduction, unit vector normalisation and subsequent multivariate data analysis were done with the statistical software the Unscrambler X 10.3 (Camo Software, Oslo, Norway). We performed Principle Component Analysis (PCA) using the NIPALS algorithm and cross validation, which is a common procedure for spectral data analysis.

Buchacher T., Wiesinger-Mayr H., Vierlinger K., Rüger B.M., Stanek G., Fischer M.B, & Weber V. (2014). Human blood monocytes support persistence, but not replication of the intracellular pathogen C. pneumoniae. BMC Immunology, 15, 60.

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2

Quantifying Phagosomal Acidification in Macrophages

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Phagosomal acidification was investigated using the acidotropic fluorescent probe LysoTracker® Red DND-99, which accumulates in acidic organelles. Macrophages were seeded in glass-bottom μ-Slides (Ibidi) at a concentration of 1.5 × 10⁵ cells mL⁻¹ and loaded for 2 h with 50 nM LTR in RPMI without phenol red. Medium was replaced prior to infection. Overnight cultures of Pma1-GFP C. albicans strains were regrown in YPD for 5 h at 30°C, cells were collected and washed in 1× PBS, and diluted to a concentration of 1.5 × 10⁷ cells mL⁻¹. Macrophages were infected with 10 μL of the C. albicans suspensions and incubated for 1 h at 37°C, 5% CO₂. Cells were fixed in paraformaldehyde, washed with 1× PBS, and nuclei were stained with NucBlue for visualization purposes. Images were captured and analyzed using Slidebook 6.0. To quantify the accumulation of LTR in the phagosome, we obtained the signal intensity profiles of GFP and LTR along a line extending beyond the edges of the fungal cell. We quantified the area under the curve of the LTR signal for a region of 20 pixels (2 μm) from each side of the C. albicans cell, outlined by the GFP signal. Experiments were performed in triplicate; a minimum of 30 fungal cells were analyzed per strain in each experiment.

Miramón P, & Lorenz M.C. (2016). The SPS amino acid sensor mediates nutrient acquisition and immune evasion in Candida albicans. Cellular microbiology, 18(11), 1611-1624.

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3

Synchronized U2OS Cell Imaging Assay

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1 × 10⁶ U2OS knock-in cells were seeded in a 10cm culture dish and incubated for 24 h. The culture medium was then replaced with fresh DMEM containing 2mM thymidine. After 18 h, cells were washed twice with PBS, allowed to recover for 6 h in fresh DMEM, and subjected to a second block with 2mM thymidine for 18 h.
Synchronized cells were then trypsinized, seeded into 8-well glass-bottom μ-slides (ibidi) at a density of 30,000 cells/well, and allowed to adhere for three hours. Culture medium was then exchanged with L-15 medium (Gibco) supplemented with 10% FBS and the indicated amounts of drugs. Cells were imaged every 15 min for a total of 24 h in a 3 × 3 grid per well using an Olympus IX83 epifluorescence microscope equipped with an Orca Fusion scMOS camera, a 403 air objective (NA 0.95), an X3-ZDC2 TruFocus drift compensator and an Okolab IX3-SVR stage top incubator set to 37°C. Images were quantified using custom Mathematica scripts.
To determine cell cycle stage, samples were taken immediately after synchronization and at the time point of drug addition, fixed and permeabilized with ice-cold 70% ethanol at 4°C overnight, stained with 1 μg/mL propidium iodine in PBS containing 0.1% Triton X-100 and 10 μg/mL RNase, and analyzed using a Sony SH800 flow cytometer.

Broder Schmidt H., Jaafar Z.A., Erik Wulff B., Rodencal J.J., Hong K., Aziz-Zanjani M.O., Jackson P.K., Leonetti M.D., Dixon S.J., Rohatgi R, & Brandman O. (2022). Oxaliplatin disrupts nucleolar function through biophysical disintegration. Cell reports, 41(6), 111629.

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4

Synchronized U2OS Cell Imaging Assay

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1 × 10⁶ U2OS knock-in cells were seeded in a 10cm culture dish and incubated for 24 h. The culture medium was then replaced with fresh DMEM containing 2mM thymidine. After 18 h, cells were washed twice with PBS, allowed to recover for 6 h in fresh DMEM, and subjected to a second block with 2mM thymidine for 18 h.
Synchronized cells were then trypsinized, seeded into 8-well glass-bottom μ-slides (ibidi) at a density of 30,000 cells/well, and allowed to adhere for three hours. Culture medium was then exchanged with L-15 medium (Gibco) supplemented with 10% FBS and the indicated amounts of drugs. Cells were imaged every 15 min for a total of 24 h in a 3 × 3 grid per well using an Olympus IX83 epifluorescence microscope equipped with an Orca Fusion scMOS camera, a 403 air objective (NA 0.95), an X3-ZDC2 TruFocus drift compensator and an Okolab IX3-SVR stage top incubator set to 37°C. Images were quantified using custom Mathematica scripts.
To determine cell cycle stage, samples were taken immediately after synchronization and at the time point of drug addition, fixed and permeabilized with ice-cold 70% ethanol at 4°C overnight, stained with 1 μg/mL propidium iodine in PBS containing 0.1% Triton X-100 and 10 μg/mL RNase, and analyzed using a Sony SH800 flow cytometer.

Broder Schmidt H., Jaafar Z.A., Erik Wulff B., Rodencal J.J., Hong K., Aziz-Zanjani M.O., Jackson P.K., Leonetti M.D., Dixon S.J., Rohatgi R, & Brandman O. (2022). Oxaliplatin disrupts nucleolar function through biophysical disintegration. Cell reports, 41(6), 111629.

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5

MDCK Cyst Formation Protocol

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The day before cyst inoculation, MDCK cells were split into a 10-cm dish at a confluency of 1:10. On the day of plating, plates were washed two times followed by trypsinization using 0.25% trypsin, centrifuged at 1,200 rpm followed by two washes, and resuspended in calcium- and magnesium-free PBS. 2 × 10³ cells were then plated on 100% matrigel on an eight-well glass-bottom μ-slide (Ibidi; 6 µl matrigel per well). Cells were then overlaid with 300 µl of 2% matrigel in DMEM. The cysts were grown for 4 d with a change of media on day 2. On day 4, cysts were fixed with 3.7% formaldehyde and processed for IF.

Awadia S., Huq F., Arnold T.R., Goicoechea S.M., Sun Y.J., Hou T., Kreider-Letterman G., Massimi P., Banks L., Fuentes E.J., Miller A.L, & Garcia-Mata R. (2019). SGEF forms a complex with Scribble and Dlg1 and regulates epithelial junctions and contractility. The Journal of Cell Biology, 218(8), 2699-2725.

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6

Fabrication of Geometrical Confinement Slides

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The method described previously (Le Berre et al., 2012 (link), 2014 (link)) to fabricate a geometrical confinement slide and multiwell confiner was followed with some modifications. The mold for the micropillar structures was fabricated using standard photolithography protocols on a silicon wafer using SU-8 2005 photoresist (MicroChem) spin coated onto the wafer such that the feature height = 4.4 µm, as determined by a Dektak XT Stylus Profilometer (Bruker Corporation). The mold was silanized before PDMS (GE Healthcare) slide pouring. A 9 × 9–mm coverglass was used instead of a 10-mm circular glass as a substrate for the PDMS confinement slide. Soft confinement pillars were made in a two-well glass-bottom μ-slide (ibidi) and cut into 9 × 9–mm square-bottom prisms and stuck without additional adhesive to the middle of the chamber lid. After a confinement slide was placed atop the pillar, the lid and pillar was soaked in culture medium for 1 h at 37°C, 10% CO₂ before use. During cell confinement and imaging, the lid was taped shut to maintain constant pressure on the cells.

Hatch E.M, & Hetzer M.W. (2016). Nuclear envelope rupture is induced by actin-based nucleus confinement. The Journal of Cell Biology, 215(1), 27-36.

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7

Visualizing Yeast Micro-colonies Using Fluorescent Markers

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A micro-colony of BY4741 with prototrophy restored by complementation with the fluorescent protein vectors yEpCFP_HIS (HIS3), yEpSapphire_LEU (LEU2), yEpVenus_URA (URA3) (Bilsland et al., 2013 (link)), and pRS411-GPDpr-mCherry (MET15) (Table 1) was grown for 2 nights on SM. Prior to imaging, colony was embedded in 2% agarose (Type I-B; Sigma) and gently transferred to a μ-slide glass bottom (ibidi). Cells were imaged with a DMI6000 inverted Leica SP5 confocal microscope, using a 10×/0.3 HC PL Fluotar Air objective, running LAS AF software (version 2.7.3.9723). Fluorescence for each marker was separated by excitation (CFP: 458 nm, Sapphire: 405 nm, Venus: 514 nm and mCherry: 561 nm). In our hands, the Sapphire-LEU2 was also visible under the imaging conditions used to visualise the Venus-URA3. For this reason, we removed Venus-URA3 channel from the colony image. A look-up table was applied to each channel post-acquisition to allow visualisation of the different channels together using ImageJ software.

Campbell K., Vowinckel J., Mülleder M., Malmsheimer S., Lawrence N., Calvani E., Miller-Fleming L., Alam M.T., Christen S., Keller M.A, & Ralser M. (2015). Self-establishing communities enable cooperative metabolite exchange in a eukaryote. eLife, 4, e09943.

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8

Biofilm Imaging on Scaffolds

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After overnight incubation, tissues were washed with 500 μL of PBS and placed into four-well chambered coverslip μ-Slide Glass Bottom (Ibidi). Tissues were stained with 5 μM Syto9 (Invitrogen), washed twice with PBS and turned upside down onto the cover glass. Samples were visualized as described for in vitro biofilm analysis. The scaffold surface was acquired using a 561 nm laser in reflection mode. Three Z-stacks randomly chosen at different positions in each chamber were acquired with a Z step of 0.7 microns.

Bonacorsi A., Trespidi G., Scoffone V.C., Irudal S., Barbieri G., Riabova O., Monakhova N., Makarov V, & Buroni S. (2024). Characterization of the dispirotripiperazine derivative PDSTP as antibiotic adjuvant and antivirulence compound against Pseudomonas aeruginosa. Frontiers in Microbiology, 15, 1357708.

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9

HRAS-GFP Transfection and Tipifarnib Treatment

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For HRAS-GFP transfection, cells were grown on μslide glass bottom (Ibidi). Cells were transfected with HRAS-GFP and the next day were treated with tipifarnib for 48h and the image acquisition was performed by confocal microscopy.

Gilardi M., Wang Z., Proietto M., Chillà A., Calleja-Valera J.L., Goto Y., Vanoni M., Janes M.R., Mikulski Z., Gualberto A., Molinolo A.A., Ferrara N., Gutkind J.S, & Burrows F. (2020). Tipifarnib as a Precision Therapy for HRAS-Mutant Head and Neck Squamous Cell Carcinomas. Molecular cancer therapeutics, 19(9), 1784-1796.

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Glass bottom μ slide

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9 protocols using glass bottom μ slide

Raman Spectroscopy of C. pneumoniae Infected Monocytes

Quantifying Phagosomal Acidification in Macrophages

Synchronized U2OS Cell Imaging Assay

Synchronized U2OS Cell Imaging Assay

MDCK Cyst Formation Protocol

Fabrication of Geometrical Confinement Slides

Visualizing Yeast Micro-colonies Using Fluorescent Markers

Biofilm Imaging on Scaffolds

HRAS-GFP Transfection and Tipifarnib Treatment

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