INS-1D cells were a gift from Dr. Sekine (Tokyo University) [28 (link)]. The cells were grown in 60-mm culture dishes at 37°C and 5% CO2 in a humidified atmosphere. The culture medium was RPMI 1640 (Sigma, St. Louis, MO, USA) supplemented with 10 mM glucose, 10% fetal bovine serum, 1 mM sodium pyruvate, 1 mM L-glutamine, and 50 μM 2-mercaptoethanol. For fluorescence imaging, the cells were cultured in a 35-mm glass-bottom dish (AGC Techno Glass Co., Ltd., Shizuoka, Japan) at 50% confluence 2 days before transfection. A plasmid encoding the GFP-tagged proteins was transfected into the cells using Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada). Experiments were performed within 2 days of transient transfection. We established stable transfectants from parental INS-1 cells expressing myristoylated alanine-rich C kinase substrate (MARCKS)-GFP or PKCα-GFP by G418 selection and cloning.
35 mm glass bottom dish
Manufactured by AGC Techno Glass
Sourced in Japan
The 35-mm glass-bottom dish is a laboratory equipment item designed for various microscopy applications. It features a glass bottom that allows for optical imaging and analysis of samples. The dish provides a controlled environment for cell culture, tissue samples, or other specimens that require visualization under a microscope.
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Lab products found in correlation
Fb 1365, by Biosera (2 mentions)
Penicillin, by Fujifilm (2 mentions)
Streptomycin, by Fujifilm (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Las x software, by Leica (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Fujifilm (1 mentions)
Paraformaldehyde solution, by Fujifilm (1 mentions)
Vybrant cm dii cell labeling solution, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (1 mentions)
Luminoview lv200, by Olympus (1 mentions)
D luciferin potassium salt, by Fujifilm (1 mentions)
Polyethyleneimine max mw 40 000, by Polysciences (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Cellmatrix type 1 c, by Nitta Gelatin (1 mentions)
Solis 445c, by Thorlabs (1 mentions)
5 protocols using 35 mm glass bottom dish
1
INS-1D Cell Culture and Transfection
2
Intracellular Peptide Localization in HeLa Cells
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HeLa cells were seeded (at a density of 1.5 × 105 cells/dish) in 35 mm glass bottom dish (AGC Techno Glass) and incubated for 24 h. After cell condition became stable, the cells were incubated with 0.1 µM of each peptide and 1% DMSO at 37 °C for 4 h. The cells were washed with phosphate buffered saline (PBS, FUJIFILM Wako Pure Chemical) three times and cell membrane was stained with 5 µM of Vybrant™ CM-DiI Cell-Labeling Solution (Thermo Fischer Scientific) for 5 min. The cells were washed with PBS and fixed with 4% paraformaldehyde solution (FUJIFILM Wako Pure Chemical) for 10 min. After washing with PBS three times, the cells were mounted with Prolong Diamond Antifade mountant with DAPI (Thermo Fischer Scientific). Intracellular distribution of green fluorescence from the peptide was observed using Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany) at the Support Unit for Bio-Material Analysis in RIKEN Center for Brain Science, Research Resources Division. The fluorescence intensity was adjusted with Leica LAS X software to compare the fluorescence distribution from peptides and cell membrane.
3
Bioluminescence Imaging using Luciferase
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Bioluminescence by luciferase was observed using Luminoview LV200 (Olympus, Osaka, Japan). Luminescence was acquired in MetaMorph® imaging system (Molecular Devices, Sunnyvale, CA). Pv11 cells were attached onto a 35 mm glass bottom dish (AGC Techno Glass Co., Ltd., Tokyo, Japan) using 50 µg/ml cationic polymer, Polyethyleneimine “Max” MW 40000 (Polysciences, Inc., Warrington, PA). The cationic polymer was prepared as described previously41 (link). D-luciferin potassium salt (Wako Pure Chemical Industries, Osaka, Japan) was dissolved in distilled water and kept at 4 °C in the dark until use.
4
MDCK Cell Nuclei Fluorescence Imaging
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The MDCK cells were cultured in a 35 mm glass-bottom dish (AGC Techno Glass, Shizuoka, Japan) up to the interval immediately before reaching confluence (pre-confluent) or 1.5 day after becoming confluent (post-confluent) at 37 °C with 5% CO2 in D-MEM (043-30085, Wako, Osaka, Japan) supplemented with 10% FBS (FB-1365, Biosera, France), 100 units/ml penicillin and 100 µg/ml streptomycin (168-23191, Wako, Osaka, Japan). For nuclei observation, cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS, permeabilized with 0.2% Triton X-100 for 10 min, washed again with PBS, and then stained with 5 µM NucleoSeeing (Funakoshi, Tokyo, Japan) in PBS. Images were obtained using the AMATERAS1.0 system with an excitation LED wavelength of 470 nm.
5
Imaging of Calcium Dynamics in HeLa Cells
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The HeLa cells stably expressing YC3.60 cells were cultured in a 35 mm glass-bottom dish (AGC Techno Glass, Shizuoka, Japan) coated with Cellmatrix Type I-C (Nitta Gelatin, Osaka, Japan) at 37 °C with 5% CO2 in FluoroBrite DMEM (A1896701, Thermo Fisher Scientific, Massachusetts, USA) supplemented with 10% FBS (FB-1365, Biosera, France), 4 mM GlutaMax (35050061, Thermo Fisher Scientific, Massachusetts, USA), 100 units/ml penicillin, and 100 µg/ml streptomycin (168-23191, Wako, Osaka, Japan) until the cells reached the confluence. Before imaging, the medium was replaced with FluoroBrite DMEM without FBS. Images were obtained using the AMATERAS1.0 system with an excitation LED wavelength of 445 nm (SOLIS-445C, Thorlabs, Newton, NJ) and an emission filter with a center wavelength of 540 nm (#86-366, Edmund Optics, Barrington, NJ) for imaging in the FRET channel. Two hundred frames were acquired at 5 s intervals (0.2 fps) with 500 ms exposure and at intervals of 106 ms (9.4 fps). The dish was stored in a stage-top incubator at 37 °C with 5% CO2. The intensity of the excitation light (LED, 445 nm) in the sample plane was 25.8 mW/cm 2 and 40.8 mW/cm 2 at 0.2 fps and 9.4 fps, respectively.
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