Nunc lab tek 2 chamber slide

Manufactured by Thermo Fisher Scientific

Sourced in United States, Denmark, Germany

Nunc Lab-Tek II chamber slides are a type of laboratory equipment designed to facilitate cell culture and microscopic analysis. They provide a convenient platform for seeding and culturing cells, as well as for subsequent observation and experimentation.

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124 protocols using nunc lab tek 2 chamber slide

Mitochondrial Imaging of Cellular Metabolic Effects

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MEFs were treated with fatty acids and BPA (16 h and 6 h) in Nunc Lab-Tek II Chamber Slides (ThermoFisher Scientific, Waltham, MA), followed by labeling with 300 nM Mitotracker Red (CMX ROS), a membrane potential-sensitive mitochondrial dye, for 30 minutes. Labeled cells were fixed in 10% formalin for 30 minutes, while being kept protected from light, and counterstained with DAPI to image nuclei. Cells were imaged at 600-fold magnification on an Echo Revolve microscope (Echo, San Diego, CA).

Mondal A., Burchat N, & Sampath H. (2020). Palmitate exacerbates bisphenol A toxicity via induction of ER stress and mitochondrial dysfunction. Biochimica et biophysica acta. Molecular and cell biology of lipids, 1866(1), 158816.

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Visualizing dSpCas9 Localization in HEK 293T Cells

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HEK 293T cells were transfected at 60–80% confluence in Nunc Lab-Tek II Chamber Slides (ThermoFisher Scientific) using the jetOPTIMUS transfection kit (Polyplus Transfection) with either pCDNA3.1-V5-dSpCas9 or pCDNA3.1-3xFLAG-dSpCas9. Slides were fixed with MeOH, blocked for 1 h at room temperature, and incubated under gentle orbital shaking with primary antibody overnight: either V5 Tag mouse monoclonal antibody (ThermoFisher Scientific #R960-25) at 1:3000 dilution or mouse monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich #F1804) at 1:1000 dilution. Slides were washed five times for 10 min with phosphate-buffered saline with Tween 20 (PBST), then incubated for 1 h at room temperature under gentle orbital shaking with secondary antibody: Goat anti-mouse IgG AlexaFluor 488 Superclonal Recombinant Secondary antibody (ThermoFisher Scientific #A28175) at 1:2000 dilution. Slides were washed five times for 10 min with PBST, then washed three more times with PBS before mounting overnight with 4',6-diamidino-2-phenylindole (DAPI). All antibodies were incubated with 5% BSA in 0.1% Tween-PBS. Immunofluorescence images were taken at 63x objective with a Zeiss LSM 780 confocal microscope in 5–10 slices, with maximum intensity projections across the entire image plane generated in Zeiss ZEN 2010 for figures.

Smargon A.A., Madrigal A.A., Yee B.A., Dong K.D., Mueller J.R, & Yeo G.W. (2022). Crosstalk between CRISPR-Cas9 and the human transcriptome. Nature Communications, 13, 1125.

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Immunocytochemistry for Neurotransmitter Receptors

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All materials, unless stated otherwise, were purchased from Millipore-Sigma (St. Louis, MO, USA). F-12 media, horse serum, antibiotics, Nunc™ Lab-Tek™ II Chamber Slides™ (Cat#: 154526) and RNase (Cat #: EN0531) were acquired from Thermo Fisher Scientific. Paraformaldehyde (32% aqueous solution; Cat#: 15714S) was acquired from Electron Microscopy Sciences (Hatfield, PA, USA), the proteinase K (20 mg/mL; Cat#: 501-PK) from Viagen Biotech (Los Angeles, CA, USA), and the normal donkey serum (Cat#: 017-000-121) was acquired from Jackson ImmunoResearch (West Grove, PA, USA). TH5487 was acquired from Tocris (Cat#: 6749) and ARI3 from Millipore-Sigma (St. Louis, MO, USA; Cat#: 262017).

Behrouzi A., Xia H., Thompson E.L., Kelley M.R, & Fehrenbacher J.C. (2022). Oxidative DNA Damage and Cisplatin Neurotoxicity Is Exacerbated by Inhibition of OGG1 Glycosylase Activity and APE1 Endonuclease Activity in Sensory Neurons. International Journal of Molecular Sciences, 23(3), 1909.

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Generation of Resting Macrophages from THP-1 and RAW 264.7 Cells

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THP-1 human monocytes (ATCC TIB-202) were suspended in complete RPMI media and incubated at 37°C/5% CO₂ for 24 h in presence of 50 ng/ml phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich), and then for 48 h in complete RPMI media without any stimulus to generate the resting mφs [21 (link)]. RAW 264.7 murine macrophages (ATCC TIB-71) were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 2 mmol/l glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Corning, Corning NY). THP-1 (human) or RAW 264.7 (murine) mφs were seeded in 6-well (1×10⁶ cells/well), 24-well (5×10⁵ cells/well) or 96-well (1×10⁴ cells/well) plates, or in Nunc Lab-Tek II chamber slides (1×10⁴ cells/well, Thermo Scientific, Waltham MA) and incubated for 2 h to allow the cells to adhere. Serum-free media was added, and macrophages were incubated in triplicate with microparticles (MPs) isolated from human or mouse plasma (10% plasma equivalent) or from media of Tc-infected cells (10% media equivalent). Macrophages (± MPs) were incubated for 1, 12, 24 or 48 h and cells and supernatants were stored at −80°C.

Chowdhury I.H., Koo S.J., Gupta S., Liang L.Y., Bahar B., Silla L., Nuñez-Burgos J., Barrientos N., Zago M.P, & Garg N.J. (2016). Gene Expression Profiling and Functional Characterization of Macrophages in Response to Circulatory Microparticles Produced during Trypanosoma cruzi Infection and Chagas Disease. Journal of Innate Immunity, 9(2), 203-216.

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Visualizing Endocytic Pathways in Cells

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Cells were seeded in NUNC Lab-Tek II chamber slides (ThermoScientific) and imaged in a humidified live-cell chamber equilibrated to 5% CO₂ and 37°C using an Olympus IX81 deconvolution microscope equipped with SlideBook Software (Version 5.0.0.14, Intelligent Imaging Innovations, Inc.). To monitor fluid phase endocytosis, cells were treated with 0.1 mgml⁻¹ AlexaFluor488-dextran (10 kDa; Life Technologies). To label acidic organelles, cells were incubated with LysoSensor DND-189 (1μM; Life Technologies) according to the manufacturer’s protocol. For detection of cathepsin B activity, cells were incubated with MagicRed Cathepsin B (ImmunoChemistry Technologies, LLC) per the manufacturer’s protocol.

Young M.M., Takahashi Y., Fox T.E., Yun J.K., Kester M, & Wang H.G. (2016). Sphingosine kinase 1 cooperates with autophagy to maintain endocytic membrane trafficking. Cell reports, 17(6), 1532-1545.

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Imaging Chicken Macrophage Dynamics

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Splenocytes were prepared as described above from 12‐week‐old CSF1R‐eGFP reporter transgenic birds. 0·2 × 10⁶ cells were plated on fibronectin‐coated 8‐well Nunc Lab‐Tek II Chamber slides (Thermo Fisher Scientific) and incubated at 41˚C with RPMI medium supplemented with 10% (v/v) heat‐inactivated FBS, 2 mM L‐glutamine and antibiotics (100 g/ml penicillin, 100 g/ml streptomycin) for one hour. After one‐hour, non‐adherent cells were gently washed off with RPMI and remaining adherent cells were incubated overnight in complete RPMI medium supplemented with 200 ng/ml recombinant chicken CSF1 produced as described previously [30 (link)]. The next day slides were chilled on ice for 30 minutes and stained using XCL1^AF647, anti‐chicken MHCII‐RPE and KUL01‐RPE (Table 1) in RPMI. After one hour, cells were washed in phenol red‐free RMPI medium. After staining, phenol red‐free RMPI with 10% (v/v) FBS was added to the cells and the chamber slides were placed on a microscope stage heated to 41˚C and imaged using a Zeiss LSM 710‐inverted microscope.

Wu Z., Hu T., Chintoan‐Uta C., Macdonald J., Stevens M.P., Sang H., Hume D.A., Kaiser P, & Balic A. (2021). Development of novel reagents to chicken FLT3, XCR1 and CSF2R for the identification and characterization of avian conventional dendritic cells. Immunology, 165(2), 171-194.

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Immunofluorescence and Confocal Microscopy Protocol

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Immunofluorescence staining and confocal microscopy were performed as described previously (10 (link),18 (link),19 (link)). Briefly, cells were grown and drug treated on chamber slides (Thermo Scientific™ Nunc™ Lab-Tek™ II Chamber slides) followed by fixation with 4% paraformaldehyde for 10 min at room temperature. Primary antibodies against PRMT5, γH2AX and XRCC1 were detected using anti-rabbit or anti-mouse IgG secondary antibodies labeled with Alexa 488/568 (Invitrogen). Cells were mounted in anti-fade solution with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) and examined under Leica TCS SP8 confocal laser-scanning microscope (Germany) with a 63×/1.4 NA oil objective. Images were collected and processed using the Leica software and sized in Adobe Photoshop 7.0. The γH2AX intensity per nucleus was determined with Adobe Photoshop 7.0 by measuring the fluorescence intensities normalized to the number of cell count (10 (link),18 (link),19 (link)).

Rehman I., Basu S.M., Das S.K., Bhattacharjee S., Ghosh A., Pommier Y, & Das B.B. (2018). PRMT5-mediated arginine methylation of TDP1 for the repair of topoisomerase I covalent complexes. Nucleic Acids Research, 46(11), 5601-5617.

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Immunofluorescence Assay for GLI1

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SKGT4 with and without siRNA transfection were seeded into 4-well Nunc Lab-Tek II Chamber Slides (Thermo Fisher Scientific, Waltham, MA). After reaching 90% confluence, the cells were fixed in acetone at −20 °C for 7 minutes, washed three times with 1xPBS, and then incubated with primary antibodies for GLI1. Subsequently, the cells were incubated with AlexaFluor goat anti-rabbit secondary antibodies. The cells were counterstained with Hoechst 33342 (Thermo-Fisher Scientific), embedded with paraffin, and observed with a Zeiss Axio Observer Inverted Microscope (Zeiss, Oberkochen, Germany) for fluorescent imaging.

Ng C.K., Ma K., Cheng Y., Miyashita T., Harmon J.W, & Meltzer S.J. (2019). Krüppel-like Factor 5 Promotes Sonic Hedgehog Signaling and Neoplasia in Barrett's Esophagus and Esophageal Adenocarcinoma. Translational Oncology, 12(11), 1432-1441.

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Lipid Staining in TC32 Cells

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TC32 cells were plated and treated in Nunc Lab-Tek II Chamber Slides (Thermo Scientific). Cells were washed with PBS, fixed for 30 minutes, and then washed with distilled water. Cells were incubated with isopropanol for 3 minutes and a working solution (filtered 3:2 Oil Red O (Sigma) to deionized water) of Oil Red O for 10 minutes. After aspirating the Oil Red O solution, the slides were briefly stained with hematoxylin solution (Sigma) and imaged using an Aperio scanning microscope (Leica, Buffalo Grove, IL).

Harlow M.L., Maloney N., Roland J., Guillen Navarro M.J., Easton M.K., Kitchen-Goosen S.M., Boguslawski E., Madaj Z.B., Johnson B.K., Bowman M.J., D’Incalci M., Winn M.E., Turner L., Hostetter G., Galmarini C.M., Aviles P, & Grohar P.J. (2016). Lurbinectedin inactivates the Ewing sarcoma oncoprotein EWS-FLI1 by redistributing it within the nucleus. Cancer research, 76(22), 6657-6668.

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SARS-CoV-2 Infection Inhibition Assay

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The day before infection, Nunc LabTek II chamber slides (Thermo Scientific, Waltham, Massachusetts) were seeded with 2.5 × 10⁴ cells/chamber. On the day of infection, chambers were treated with dilutions of ATN-161 in complete DMEM with 2% fetal bovine serum for 1 h before infecting with SARS-CoV-2 at a multiplicity of infection of 0.01, which was chosen for ease of visibility of viable cells versus cytopathic cells under microscopy. Slides were placed in a 37°C 5% CO₂ incubator for 24 h before imaging via phase contrast using an EVOS XL inverted microscope (Thermo Scientific).

Beddingfield B.J., Iwanaga N., Chapagain P.P., Zheng W., Roy C.J., Hu T.Y., Kolls J.K, & Bix G.J. (2020). The Integrin Binding Peptide, ATN-161, as a Novel Therapy for SARS-CoV-2 Infection. JACC: Basic to Translational Science, 6(1), 1-8.

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