μ slide 8 well glass bottom slides

Manufactured by Ibidi

Sourced in United Kingdom

The μ-Slide 8 Well Glass Bottom slides are a laboratory equipment product designed for cell culture and microscopy applications. The slides feature eight individual wells with a glass bottom, providing a clear optical surface for imaging and analysis.

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10 protocols using μ slide 8 well glass bottom slides

Mitochondrial Morphology Analysis of Th17 Cells

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After FACS-sorting, Th17 cells were cultured in μ-Slide 8 well glass bottom slides (ibidi GmbH) and incubated for 30 min at 37°C with MitoTracker™ Red CMXRos (100 nM, ThermoFisher). After two rinses with PBS, cells were fixed with paraformaldehyde 3.7% for 15 min. DAPI (1 μg/mL, Sigma) and Alexa Fluor™ 488 Phalloidin (150 nM, ThermoFisher) were used for nuclear and actin staining, respectively.
Z-stack images were acquired using a Leica TCS SP8 (Leica Microsystems, Germany) confocal microscope and spatial deconvolution and 3D surface-rendered images were carried out with Huygens Software (SVI, The Netherlands). Mitochondrial morphology was analysed using the macro MiNa in Fiji software (Schindelin et al., 2012 (link), Valente et al., 2017 (link)).

Omenetti S., Bussi C., Metidji A., Iseppon A., Lee S., Tolaini M., Li Y., Kelly G., Chakravarty P., Shoaie S., Gutierrez M.G, & Stockinger B. (2019). The Intestine Harbors Functionally Distinct Homeostatic Tissue-Resident and Inflammatory Th17 Cells. Immunity, 51(1), 77-89.e6.

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Visualizing hMSC Morphology with CoBG

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To visualise hMSC morphology in the presence of CoBG-conditioned medium, hMSCs were stained for filamentous actin (F-actin). In brief, passage 5 hMSCs were seeded at a density of 4000 cells/cm² on μ-Slide 8 Well Glass Bottom slides (ibidi^®, UK) and cultured in CoBG-conditioned or control medium. Control medium (α-MEM) supplemented with 100 μM CoCl₂ was used as positive control. After 1, 4 and 7 days of culture, cells were fixed with 3.7% (v/v) paraformaldehyde in PBS for 20 min. Cells were permeabilised in 0.2% (v/v) Triton-X 100 in PBS for 15 min and blocked with 1% (v/v) goat serum for 30 min. The actin cytoskeleton was stained with phalloidin-Alexa 488 (Life technologies, UK) and the nuclei were stained with DAPI. Imaging with fluorescent microscope (EVOS^® FL Cell Imaging System; Life Technologies) was conducted immediately after staining.

Littmann E., Autefage H., Solanki A.K., Kallepitis C., Jones J.R., Alini M., Peroglio M, & Stevens M.M. (2018). Cobalt-containing bioactive glasses reduce human mesenchymal stem cell chondrogenic differentiation despite HIF-1α stabilisation. Journal of the European Ceramic Society, 38(3), 877-886.

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TIRFM Imaging of AEC Attachment

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For TIRFM, AECs were grown on fibronectin-coated μ-slide 8 well glass bottom slides (Ibidi) for 4 days and stained with 5 μl/ml Vybrant DiO cell labeling solution (Thermofisher) for 10 min at 37 °C. Life imaging was performed in the imaging facility of the Molecular Biophysics group, HU Berlin on a Confocal Laser Scanning Microscope (Olympus FV-1000MPE) equipped with a TIRFM upgrade at 37 °C and 5% CO₂ using a 63x oil objective and a cooled CCD camera. Images were taken at indicated time points and analysed using FijI. Particle analysis was performed from binary images after thresholding (method Isodata). Total intensity of attachment from all detected particles was measured.

Kostadinova E., Chaput C., Gutbier B., Lippmann J., Sander L.E., Mitchell T.J., Suttorp N., Witzenrath M, & Opitz B. (2016). NLRP3 protects alveolar barrier integrity by an inflammasome-independent increase of epithelial cell adherence. Scientific Reports, 6, 30943.

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Evaluating SAFE-Modified Antibody Staining

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To verify that staining accuracy is not affected by the bioorthogonally cleavable linker, SAFE-modified antibodies were compared to conventionally labeled controls. A representative panel of anti-CD11b (MB488), CD45 (AF555), CD8 (MB594), and Ly6G (AF647) antibodies were modified with individual SAFE probes, as (specified). In parallel, control antibodies in a non-overlapping color/channel were prepared via conventional activated ester labeling. AZDye 555 NHS Ester (Fluoroprobes, 1166–5) was used to label anti-CD11b, while AFDye 488 TFP ester (Fluoroprobes, 1026–5) was used to label the parent anti-CD45, CD8, and Ly6G antibodies. Control antibodies with a degree of labeling (DOL) of 2–3 fluorophores/antibody were prepared to match the DOL of SAFE modified antibodies. Control and SAFE modified antibodies (each at 5 μg/mL) were then premixed and co-incubated with mouse splenocytes for 10 minutes at room temperature. After washing three times with PBS (5–10 sec each), the stained cells were imaged on μ-Slide 8 Well Glass Bottom slides (Ibidi, 80826) using a DeltaVision inverted fluorescence microscope (Supplementary Fig. 5).

Ko J., Wilkovitsch M., Oh J., Kohler R., Bolli E., Pittet M.J., Vinegoni C., Sykes D.B., Mikula H., Weissleder R, & Carlson J.C. (2022). Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes. Nature biotechnology, 40(11), 1654-1662.

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Visualizing hMSC Morphology under CoBG Influence

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To visualise hMSC morphology in the presence of CoBG-conditioned medium, hMSCs were stained for filamentous actin (F-actin). In brief, passage 5 hMSCs were seeded at a density of 4,000 cells/cm² on μ-Slide 8 Well Glass Bottom slides (ibidi^®, UK) and cultured in CoBG-conditioned or control medium. Control medium (α-MEM) supplemented with 100 μM CoCl₂ was used as positive control. After 1, 4 and 7 days of culture, cells were fixed with 3.7% (v/v) paraformaldehyde in PBS for 20 min. Cells were permeabilised in 0.2% (v/v) Triton-X 100 in PBS for 15 min and blocked with 1% (v/v) goat serum for 30 min. The actin cytoskeleton was stained with phalloidin-Alexa 488 (Life technologies, UK) and the nuclei were stained with DAPI. Imaging with fluorescent microscope (EVOS^® FL Cell Imaging System; Life Technologies) was conducted immediately after staining.

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Haploid Cell Line CRISPR Editing

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Baz2B-KO (HZGHC000523c005, 16 bp deletion in the coding exon in Baz2B was edited by CRISPR-Cas9) and its parental Hap1 cell lines were purchased from Horizon Discovery (Cambridge, UK). Cells were cultured in IMDM (from Gibco™, serum-free medium) supplemented with l-glutamine, phenol red indicator, and 10% FCS. Cells were typically maintained at 40–90% confluence and passaged every 2–3 days. The two cell lines were treated separately.
An inducible U2OS cell line expressing BAZ2B-halo was generated by cloning the BAZ2B gene (UniProt: Q9UIF8, codon optimised by Genescript) into the pEBTet GW_Halo plasmid vector, transfecting and selecting plasmid-containing cells in McCoys 5A medium (ThermoFisher #16600082) with 10% FBS (BioSELL, #S0615) supplemented with 1 mM sodium pyruvate (Sigma, #S8636), 1% penicillin-streptomycin (Sigma #P0781) and 1 μg/ml puromycin (Sigma, #P9620) in a humidified 5% CO2 incubator at 37°C for 1–2 weeks (64 (link),65 (link)). For imaging, the cells were grown in μ-Slide 8-well glass bottom slides (Ibidi) for 48 h and gene expression was induced with 0.1 μg/ml doxycycline for a further 24 h. The BAZ2B-halo was stained by incubation with 100 nM JF646-halo for 1 h and the cells were fixed with 4% PFA.

Breindl M., Spitzer D., Gerasimaitė R., Kairys V., Schubert T., Henfling R., Schwartz U., Lukinavičius G, & Manelytė L. (2023). Biochemical and cellular insights into the Baz2B protein, a non-catalytic subunit of the chromatin remodeling complex. Nucleic Acids Research, 52(1), 337-354.

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Cell Culture Optimization for Imaging

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The A549 (CCL-185) and A431 (CRL-1555) cell lines were purchased from the American Tissue Culture Collection (ATCC) and used in method development experiments. A549 cells were passaged in F-12K (ATCC, 30–2004) with 10% FBS and 1% penicillin/streptomycin according to the specifications from ATCC. Cells were first grown in a 150 mm cell culture dish and then seeded on μ-Slide 8 Well Glass Bottom slides (Ibidi, 80826) for imaging. After 24–48 hours, confluency was assessed and cells were stained and imaged.

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Microwell Fabrication for Non-Adherent Cells

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The microwells used to culture and/or image nonadherent cells were fabricated at the Soft Materials Cleanroom (SMCR), Harvard Center for Nanoscale Systems (CNS). Each microwell array consists of 1681 total wells. The microwells (W: 60 μm, H: 80 μm) were made using soft lithography with SU-8 3050. Different widths were tested (60, 80, 100 μm) and 60 μm was chosen, a dimension that demonstrated no detectable cell loss throughout multiple experimental steps (e.g., media change, incubation, washing, culture, etc.). Polydimethylsiloxane (PDMS) was spin coated on a patterned wafer, cured in 65 °C for an hour, peeled off from the wafer, and then transferred to μ-Slide 8 Well Glass Bottom slides (Ibidi) for cell culture and imaging.

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Live-cell Imaging of EGFR Dynamics

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A549 cells were cultured in μ-Slide 8 Well Glass Bottom slides (Ibidi) for 24–48 hours before imaging. After 24–48 hours, cells were stained with 5 μg/mL of SAFE conjugated (AF647) anti-EGFR antibody for 10 mins at RT, and then rinsed with three successive washes of PBS, culminating with addition of live cell imaging solution (Thermo Fisher, A14291DJ). After image acquisition, quenching was performed by incubating the stained live cells with BHQ3-N-Tz (2) at 600 nM. Fluorescent images were acquired after staining, 1 min after quenching/scission, and 24 hours after scission. After 24 hours, cells were stained with calcein AM (Thermo Fisher, C1430) and SYTOX Red (Thermo Fisher, S34859) to check cell viability. A DeltaVision inverted fluorescence microscope was used to acquire all the fluorescent images in this study.

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Quantifying Mitochondrial Dynamics in Microglia

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Freshly sorted microglia were cultured in μ-Slide 8 well glass bottom slides (ibidi GmbH) and incubated for 30 min at 37°C with MitoTracker™ Red CMXRos (100 nM, ThermoFisher). After two rinses with PBS, cells were fixed with 3.7% paraformaldehyde for 15 min. DAPI (1 μg/mL, Sigma) and Alexa Fluor™ 488 Phalloidin (150 nM, ThermoFisher) were used for nuclear and actin staining. Z-stack images were acquired and mitochondrial area was quantified in Fiji software.

He D., Xu H., Zhang H., Tang R., Lan Y., Xing R., Li S., Christian E., Hou Y., Lorello P., Caldarone B., Ding J., Nguyen L., Dionne D., Thakore P., Schnell A., Huh J.R., Rozenblatt-Rosen O., Regev A, & Kuchroo V. (2022). Disruption of the IL-33-ST2-AKT signaling axis impairs neurodevelopment by inhibiting microglial metabolic adaptation and phagocytic function. Immunity, 55(1), 159-173.e9.

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