Primary EO cells were isolated from the lower incisors of Sprague-Dawley rats (100 to 120 g). Secretory and maturation EO cells were isolated as previously described using a molar reference line (38 (link), 39 (link)). EO cells were digested with Liberase (0.25 mg/ml; Roche) for 30 min at 37°C, washed in Hanks’ balanced salt solution, and plated onto CellTak-coated (Corning) coverslips in X-Vivo15 medium (Lonza) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/ streptomycin, and 1% glutamine. Isolated EO cells were used within 24 hours after dissection. LS8 cells are an immortalized murine- derived enamel cell line (37 (link)). LS8 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C with 5% CO2.
Cell tak coated
Manufactured by Corning
Sourced in United States
Cell-Tak-coated is a cell adhesive product developed by Corning. It is designed to facilitate the attachment of cells to various substrates used in cell culture applications. The product is a protein-based solution that can be applied to the surface of cell culture vessels, coverslips, or other laboratory equipment to enhance cell attachment and growth.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp8 confocal laser scanning microscopy system, by Leica (2 mentions)
Liberase, by Roche (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
2 7 dichlorofluorescin diacetate dcf da, by Thermo Fisher Scientific (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Celltracker deep red, by Thermo Fisher Scientific (1 mentions)
A1r storm, by Nikon (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Xf cell mito stress test kit, by Agilent Technologies (1 mentions)
Xfe96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Seahorse xf cell mito stress test kit, by Agilent Technologies (1 mentions)
Seahorse xf24 extracellular flux analyser, by Agilent Technologies (1 mentions)
Seahorse xf glycolytic rate assay kit, by Agilent Technologies (1 mentions)
Glass bottom dishes, by Ibidi (1 mentions)
Dcf da, by Thermo Fisher Scientific (1 mentions)
Seahorse xfe96 analyzer, by Agilent Technologies (1 mentions)
7 protocols using cell tak coated
1
Isolation and Culture of Enamel Organ Cells
2
Confocal Imaging of Cellular Peroxide
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Confocal microscopy Imaging was performed as described in [24 (link)]. Briefly, cells were cytospun on fibronectin-coated (Cell-tak-coated (Corning, New York, USA)) 35-mm glass-bottom dishes (Ibidi, Gräfelfing, Germany)] and incubated for 20 min at 37 °C with 5 μM DCF-DA (2,7-dichlorofluorescin diacetate) (Molecular Probes, Eugene, Oregon, USA) to detect cellular peroxide. Stained cells were washed with PBS and examined by a Leica (Wetzlar, Germany) TCS SP8 confocal laser scanning microscopy system utilizing appropriate excitation laser beams (images collected using a 60X objective (1.4 NA). Acquisition, storage, and analysis of data were performed with LasX software from Leica or ImageJ 1.48 (Wayne Rasband, NIH, Bethesda, Maryland, USA).
3
Imaging T-cell Mediated Cytotoxicity
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Daudi cells were labelled with CellTracker Deep Red (Invitrogen, Carlsbad, California, USA), according to the manufacturer's instructions, seeded on a Cell-Tak-coated (Corning) chamber slide (Thermo Scientific Nunc), incubated at 37 °C for 30 min, followed by incubation with 1 µg/mL DuoBody-CD3xCD20 and T cells (1:1 ratio Daudi:T cells, 24 h). Cells were fixed in 2% formaldehyde at RT for 10 min and stained for CD69 or bound DuoBody-CD3xCD20 at RT for 45 min. Intracellular staining of perforin was performed after the cells were permeabilized with Cytofix/Cytoperm solution (BD Biosciences) at 4 °C for 20 min (antibodies listed in Suppl. Table 1). All samples were washed with PBS and mounted using vectashield (with DAPI, Vector Laboratories). Images were taken with a confocal laser scanning microscope Nikon A1R/STORM and a 60x objective (Apo 60x Oil λS DIC N2). Acquired images were processed with ImageJ [17] (link).
4
Metabolic Profiling of Primary T Cells
5
Extracellular Flux Analysis of T Cells
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OCR and glycoPER were measured using a Seahorse XF24 Extracellular Flux Analyser (Seahorse Bioscience, Agilent Technologies, Santa Clara, CA). A Seahorse XF Cell Mito Stress Test Kit (103015-100; Seahorse Bioscience) and Seahorse XF Glycolytic Rate Assay Kit (103344-100; Seahorse Bioscience) were also used. T cells (3×105/well) were seeded in 24-well Cell-Tak-coated (22.4 μg/ml, Corning) Seahorse culture plates for analysis. For OCR measurement, 1.5 μM oligomycin, 0.5 μM FCCP and 0.5 μM antimycin-A/rotenone were added sequentially. To measure the glycoPER and mitoOCR/glycoPER ratio, 0.5 μM Rot/AA and 50 mM 2-DG were added sequentially.
6
Mitochondrial Membrane Potential and Oxidative Stress Imaging
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Cells were cytospun on fibronectin-coated (Cell-tak-coated (Corning, New York, USA)) 35-mm glass-bottom dishes (Ibidi, Gräfelfing, Germany)] and incubated for 20 min at 37 °C with 100 nM Tetramethylrhodamine, ethyl ester (TMRE, Life Technologies, Carlsbad, CA, USA) to detect the mitochondrial membrane potential or with 5 μM DCF-DA (2,7-dichlorofluorescin diacetate) (Molecular Probes, Eugene, Oregon, USA) to detect cellular peroxide. Stained cells were washed with PBS and examined by a Leica (Wetzlar, Germany) TCS SP8 confocal laser scanning microscopy system utilizing appropriate excitation laser beams (images collected using a 60X objective (1.4 NA)). Acquisition, storage, and analysis of data were performed with LasX software from Leica or ImageJ 1.48 (Wayne Rasband, NIH, Bethesda, Maryland, USA).
7
Measuring Cellular Oxygen Consumption
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Oxygen consumption rate was measured using the Seahorse XFe96 analyzer (Agilent) according to the manufacturer's instructions. Briefly, 50,000 cells were plated in each well of a 96-well Seahorse plate. Adherent cells were plated the night prior to the assay, and non-adherent cells (603 and 667) were plated on Cell-Tak-coated (Corning) plates on the day of the assay according to the manufacturer's instructions. At least 6 hours prior to the assay, the assay cartridge was hydrated in water in a non-CO 2 incubator at 37°C. Cells were incubated in a non-CO 2 incubator for 45 minutes. 10 mM NAC pH 7.4 was loaded into appropriate wells of the flux pack and placed into the XFe96 analyzer. The plate containing cells was subsequently loaded into the XFe96 analyzer, and OCR was analyzed using Seahorse Wave software.
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