During apoptosis, cleavage of genomic DNA yields double-stranded DNA breaks that are identifiable by labeling the free 3′-OH termini with modified nucleotides in an enzymatic reaction; the TUNEL assay is based on this principle. In brief, PC12 cells were grown on confocal dishes (NEST, Wuxi, China) in DMEM-F12 for 24 h, and then pretreated with the compounds of interest alone or in combinations for 1 h. The pretreated cells were then exposed to 20 µM Aβ25−35 for 24 h at 37 °C and 5% CO2. The cells were washed twice with PBS and then fixed with 4% paraformaldehyde (PFA) at room temperature (24 °C). The fixed cells were washed twice with PBS buffer, treated with 0.1% TritonX-100 to permeate cells, and then washed twice with PBS. Next, TUNEL reaction mixture (Beyotime Institute of Biotechnology) was added to the cells, which were then incubated for 60 min at 37 °C in a dark, humidified environment. Negative controls were prepared with equal volumes of labeling solution. After this incubation, the confocal dishes (NEST) were rinsed twice with PBS. Finally, the cells were analyzed using a confocal Laser Scanning Biological microscope FV1000 (Olympus, Tokyo, Japan) at an excitation wavelength of 488 nm.
Confocal dishes
Manufactured by NEST Biotechnology
Sourced in China, United States
Confocal dishes are specialized cell culture dishes designed for use with confocal microscopy. They feature a thin, optically transparent bottom that allows for high-resolution imaging of cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Laser scanning biological microscope fv1000, by Olympus (2 mentions)
Tcs sp8, by Leica camera (2 mentions)
Zen software, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Leica sp5 confocal microscope, by Leica Microsystems (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Culture plates, by Corning (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Urea, by Maclin Biochemical Technology (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Goat serum, by Beyotime (1 mentions)
Hoechst 33342 staining solution, by Beyotime (1 mentions)
33 protocols using confocal dishes
1
Quantifying Apoptosis-Induced DNA Breaks
2
Transient Transfection of HEK293T Cells
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HEK293T cells were cultured in DMEM (Hyclone) containing 10% fetal bovine serum, 4.5 g/L glucose, 4.0 mM L-glutamine and sodium pyruvate. Cells were maintained at 37 °C in humidified atmosphere of 5% CO2. For transfection, HEK293T cells were seeded in culture plates (CORNING) or confocal dishes (NEST) for 20 h and transfected with plasmids using Lipofectamine 2000 (Invitrogen) following the protocol of manufacturer. Mitochondria were visualized by incubating cells with MitoTracker Deep Red (100 nM) (Molecular Probes: M22426) for 30 min at 37 °C with 5% CO2.
3
Imaging co-cultured cells with fluorescent markers
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MitoTracker Deep Red labeled T24 cells were co-cultured with RT4 cells at a 1:1 ratio for 24 h in Confocal dishes (NEST Biotechnology Co., LTD, Wuxi, China, #801002), and then fixed with 4% paraformaldehyde (PFA). F-actin and nuclei were stained with Actin-Tracker Green and DAPI, respectively. Cells were observed under a fluorescence microscope (OLYMPUS-BX53) and Laser Confocal Microscope (LCM) (Leica SP8 STED), and images were captured by DP73-CellSens Entry and the Leica LCS SP8 STED system.
4
Multiplex Protein Detection Assay
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Urea, arginine, and glycerol were purchased from MACKLIN; Tris and ethylenediaminetetraacetic acid (EDTA) were purchased from Biofroxx; sodium chloride was purchased from Guangdong Guanghua Sci-Tech Co., Ltd.; dithiothreose alcohol, 3, 3', 5, 5' - tetramethylbenzidine (TMB) substrate for ELISA, ELISA stopping solution, glue-activated horseradish peroxidase labeling kit, and Triton X-100 were purchased from Solarbio; Freund’s complete adjuvant, Freund’s incomplete adjuvant, HAT, and HT were purchased from Sigma; goat anti-mouse IgG (H&L) horseradish peroxidase (HRP) was purchased from Sango Biotech; fetal bovine serum and RPMI 1640 medium were purchased from ThermoFisher Scientific; PBS was purchased from GBCBIO Technologies; saturated ammonium sulfate solution was purchased from Leagene; PAGE gel quick preparation kit was purchased from ATGene; and non-reducing loading buffer and reducing loading buffer were purchased from GBCBIO Technologies, Inc. Confocal dishes were purchased from NEST Biotechnology Co., and 4% paraformaldehyde, goat serum, Hoechst 33342 staining solution, and anti-fluorescence quenching sealer were purchased from Beyotime. Fluorescein (FITC)-conjugated goat anti-mouse IgG (H + L) was purchased from Proteintech.
5
Apoptosis Detection in Cardiovascular Cells
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An in situ cell death detection kit (terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling [TUNEL] assay kit, Roche Applied Science, 11767291910, Basel, Switzerland) was used to assess cell apoptosis in vivo and in vitro. Apoptotic cells in the sections of mouse myocardial tissues were stained according to the manufacturer's instructions, and slides were counterstained for endothelial cells using an antibody against CD31. HUVECs were grown in confocal dishes (NEST, Wuxi, China) before TUNEL staining. The experiment was carried out according to the manufacturer's instructions. 4′,6-diamidino-2-phenylindole (DAPI, Abcam, ab104139, Cambridge, UK) was used to stain the cell nuclei. Images were captured by LEICA TCS SP5 MP (Leica, Weztlar, Germany).
6
Immunostaining Protocol for HEK293T Cells
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HEK293T and HEK293T-i-3CS-GLuc2 cells were seeded in confocal dishes (NEST). At 24 hours post seeding, cells were washed, fixed with 4% formaldehyde, permeabilized with 2% Triton-X100, blocked with PBS buffer containing 2% BSA and 5% normal goat serum (Beyotime), and immunostained with anti-Flag monoclonal antibodies and FITC-labeled secondary antibodies. After washing, cell nuclei were counterstained with Hoechst 33258 (Beyotime) and visualized on a PerkinElmer UltraView VOX system using a Nikon Ti microscope.
7
Evaluating DNMT1 Targeting by Apt. #9
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To confirm that Apt. #9 can target DNMT1 protein in living cells, we used the transient transfection method to increase DNMT1 expression in HEK 293T cell. Briefly, 1 × 105 cells were seeded in confocal dishes (NEST Biotechnology Co. Ltd) and cultured for 24 hrs. Followed, the cells were transfected with the DNMT1 plasmid (pKanCMV_mRuby_DNMT1, full length) using the TransIT®-2020 Transfection Reagent (Mirus Bio Company) according to the protocol provided by the manufacturer. The transfected cells were cultured for another 24 hrs, before incubation with Apt. #9 or ssDNA #12 and then inspection by confocal laser scanning microscopy.
8
Visualizing DNA Aptamer Binding in HeLa Cells
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HeLa cells (HeLa ATCC® CCL-2™) were cultured in RPMI-1640 (contain 10% FBS, 1% penicillin and 1% streptomycin). Before the confocal imaging experiment, cells were seeded at desired concentrations in confocal dishes (NEST Biotechnology Co. Ltd). After cultured for 24 hrs, they were washed twice with 1× PBS, then incubated with 3 μM Apt. #9 or the non-binding ssDNA #12, both labeled with Alexa Fluor488, for 3 hrs at 37°C. Two washes were applied before the live cells were imaged by the LSM 880 with Airyscan Microscope (Zeiss). The dye labels on the DNA strands were excited at 488-nm by an argon ion laser and the emission was collected at 500–600 nm. Nuclei were stained by Hoechst 33342 which was excited with a 355 nm UV laser and the emission was collected at 400–480 nm. For the transfection sample, mRuby was excited at 561 nm and the emission was collected at 570–650 nm. These images were stacked and reconstructed by ZEN software. For co-localization analysis, the high resolution Airyscan model was employed for imaging; and the images were analyzed using the colocalization analysis package plugin of ImageJ.
9
Lysosomal Trafficking in 4T1 Cells
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4T1 cells in logarithmic phase were seeded in confocal dishes (NEST Biotechnology, Wuxi, China) at a density of 105 cells per well and incubated overnight in a three-gas incubator. CTP/CDDP/C6 containing 0.1 μg/mL C6 was then added to the media and co-incubated with 4T1 cells for 1 h or 3 h. Subsequently, the medium was abandoned and the Lyso-Tracker Red working solution (70 nM) preheated at 37 ℃ was added to the confocal dishes for an additional incubation of 2 h. Then the cells were washed with PBS for 3 times, fixed with 4% paraformaldehyde for 15 min, stained with DAPI (10 μg/mL) for 10 min and observed under a Nikon Eclipse Ti confocal laser scanning microscope (CLSM, Tokyo, Japan).
10
BMDC Internalization of Cy-5-CpG and CNPs
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BMDCs were established by culturing bone marrow cells isolated from the femurs of C57BL/6 mice in RPMI 1640 (Gibco, USA) medium supplemented with 10% FBS (Gibco, USA), 50 µM β-mercaptoethanol (Sigma-Aldrich, USA), 1% penicillin/streptomycin (Gibco, USA), 20 ng/ mL GM-CSF and 5 ng/mL IL-4 (Peprotech, USA). On day 3 the media were all replaced and on day 6 the media were half replaced. Collected immature 2.5 × 105 of BMDCs were put into confocal dishes (NEST, China) for 24 h at 37℃ with 5% CO2 on day 7. BMDCs were then incubated with Cy-5-labeled CpG ODN, and CNPs for 24 h. After washing with PBS for three times, cells were fixed by 4% paraformaldehyde for 30 min followed by labeling of 4’, 6-diamidine-2’-phenylindole dihydrochloride (DAPI, Thermo Fisher, USA) for 10 min. The cells were subsequently washed 2 times with PBS and photographed by using confocal laser scanning microscopic (CLSM, Carl Zeiss, USA).
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