To determine the level of intracellular Ca2+, INS-1 cells were seeded into a 12-well cell culture plate at a density of 5 × 105 cells/mL (500 μL/well). After 24 h, the medium was washed twice with Krebs-Ringer bicarbonate HEPES buffer (KRBB, 4.8 mM KCl, 129 mM NaCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 10 mM HEPES, 5 mM NaHCO3, and 0.1% BSA, pH 7.4) and 2.8 mM glucose. Cells were allowed to starve in new KRBB for 2 h. Then the buffer was removed and the cells were treated with α-mangostin (5 μM). Next, high glucose concentrations (16.7 mM) were added to the culture medium. After 1 h, 24 h, and 48 h, cells were stained with 2 µM Fluo-4 AM (Invitrogen, Eugene, OR, USA), a fluorescent indicator for Ca2+. Fluorescent Ca2+ images were captured digitally using a fluorescent microscope equipped with a cooled CCD camera (Olympus, Tokyo, Japan).
Cooled ccd camera
Manufactured by Olympus
Sourced in Japan
The Cooled CCD Camera is a high-performance imaging device designed for scientific and research applications. It features a charge-coupled device (CCD) sensor that is cooled to reduce thermal noise, enabling the capture of high-quality, low-noise images. The camera's core function is to provide precise and sensitive image acquisition for a variety of scientific and analytical tasks.
Automatically generated - may contain errors
Lab products found in correlation
Axioplan 2, by Zeiss (3 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Celltracker blue cmac, by Thermo Fisher Scientific (1 mentions)
Axioskop 40, by Zeiss (1 mentions)
Tsa system, by PerkinElmer (1 mentions)
Bz x710, by Keyence (1 mentions)
5 protocols using cooled ccd camera
1
Ca2+ Imaging of INS-1 Cells
2
Fluorescent Labeling of Yeast Cells
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Cells grown in liquid YPD were collected at exponential phase, washed with H2O, and then with 10 mM HEPES buffer pH 7.4, containing 5% D-glucose, resuspended at a density of 106 cells/ml in the same HEPES buffer, containing glucose and 200 μM CellTracker Blue CMAC (Thermo Scientific). Cells were incubated for 30–45 min at room temperature and visualized by fluorescence microscopy using an Axioskop 40 (Zeiss, Oberkochen, Germany) with a cooled CCD camera (Olympus Corporation, Tokyo, Japan). Images were assembled in Photoshop (Adobe) with only linear adjustments.
3
Hoechst Staining Apoptosis Assay
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Cell death with the typical morphological features of apoptosis including pyknotic nuclei was assessed by staining cell nuclei with Hoechst 33342 (Sigma, 2.5 μM) for 40 min in 5% CO2 at 37°C. After incubation, samples were observed using a fluorescent microscope (Axioplan2, Zeiss) with Cooled CCD Camera (DP73, Olympus), and the number of pyknotic cells in each sample was counted.
4
Immunohistochemical Analysis of Brain Sections
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Brain sections were incubated with 3% BSA in PBS containing 0.3% Triton X-100 for 30 min. Antigen retrieval was performed at 120 °C for 3 min (for CD4 and Fxop3 staining) and 20 min (for Olig2 staining) in trisodium citrate buffer (10 mM, pH 6.0) before blocking nonspecific binding sites. Then the samples were incubated with primary antibodies including mouse monoclonal antibodies directed against ED-1 (1:400, Bio-Rad, Cat# MCA341R), CD 4 (1:500, Bio-Rad, Cat# MCA372G) and STEM121 (1:500, Takara Bio, Cat# Y40410), a rat monoclonal antibody directed against Foxp3 (1:500, Invitrogen, Cat# 13-5773-82) and a rabbit polyclonal antibody directed against Olig2 (1:200, IBL, Cat# 18953) at 4 °C overnight. For ED-1, STEM121, and Olig2 staining, after washing with PBS, the sections were incubated with fluorophore-conjugated secondary antibodies for 2 h at room temperature. For CD 4 and Foxp3 detection, the samples were stained by TSA system (Perkin Elmer) to amplify the positive tyramide signal. Nuclei were stained with Hoechst 33342 (1µg/ml, Sigma, Cat# B2261) for 1 h at room temperature. Following rinsing by PBS, the sections were mounted with coverslips using Vectashield mounting medium. All stained brain sections were observed using a fluorescent microscope (Axioplan2, Zeiss) with Cooled CCD Camera (DP73, Olympus) or a fluorescence microscope (BZ-X710, Keyence, Japan).
5
Cell Proliferation Assessment via BrdU Staining
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To assess cell proliferation, bromodeoxyuridine (BrdU; 10 μM) (Roche Diagnostics) was added to the cultures for the last 4 h of culture, followed by fixation and staining with rat monoclonal BrdU antibody (abcam; 1:1000). Propidium iodide (Sigma, 2 μg/ml) was used for nuclear staining. Samples were observed using a fluorescent microscope (Axioplan2, Zeiss) with Cooled CCD Camera (DP73, Olympus), and the number of BrdU-positive cells in each sample was counted.
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