To detect the activation of β-Galactosidase as a parameter for senescence processes in CMs the β-Galactosidase activity Cell Event™ Senescence Green Detection Kit (Thermo Fisher Scientific). The staining was performed at 37°C in the absence of CO2 for 1 to 2 h. The enzyme cleavage the substrate β-galactosidase into monosaccharides in an acidic pH medium. A fluorescence product covalently interacts with intracellular proteins. Quantification of the fluorescence intensity was performed at absorption 490 nm and emission wavelength of 514 nm (three independent experiments, n = 35 CMs from each condition) was determined by the imageJ software. Then the captured images were evaluated by imageJ to analyse the senescence versus non-senescent CMs. CMs from each condition (c-CMs and SMG-CMs) were selected and the GFP intensity was quantified using the imageJ software. Then the raw intensity data were plotted.
Cellevent senescence green detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CellEvent™ Senescence Green Detection Kit is a fluorescence-based tool designed to detect and quantify senescent cells in cell culture samples. The kit utilizes a senescence-associated β-galactosidase (SA-β-gal) activity detection reagent to provide a simple and reliable method for visualizing and measuring the levels of senescent cells.
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Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (6 mentions)
Senescence detection kit, by Abcam (4 mentions)
Evos fl, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Sa β gal staining kit, by Beyotime (3 mentions)
Fv3000 confocal laser scanning microscope, by Olympus (3 mentions)
8 well chamber slide, by Ibidi (3 mentions)
Rt 157 8, by Electron Microscopy Sciences (3 mentions)
Cell event senescence green probe, by Thermo Fisher Scientific (3 mentions)
Immobilon fl pvdf membrane, by Merck Group (3 mentions)
Phosphatase inhibitor cocktail sets 1 and 2, by Merck Group (3 mentions)
Complete edta free, by Roche (3 mentions)
Bz x810 microscope, by Keyence (3 mentions)
4 chamber slides, by Merck Group (3 mentions)
Brdu flow kit, by BD (3 mentions)
Apo brdu kit, by BD (3 mentions)
44 protocols using cellevent senescence green detection kit
1
Quantifying Cardiomyocyte Senescence via β-Galactosidase
2
Senescence Detection in Engineered RPE1 Cells
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The RPE1 cells were cultured with doxycycline for 9 days, or treated with doxorubicin (500 nM) for 48 hours, followed by PBS washout and culturing for 9 days before collection. Cells were seeded into ibidi 8 well chambers one day prior to senescence detection using the CellEvent senescence green detection kit (Thermo Fisher Scientific). Samples were prepared according to the manufacturer's protocol. More than 50 cells were visualized in each cell line, with one clonal line overexpressing mCherry and two clonal lines overexpressing LmnB1. Two biological replicates were analyzed for each cell line.
3
Senescence Assay of BM-MSCs
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BM-MSCs from cell culture passage two were thawed and expanded in EM until cell culture passage four and five. After passaging by trypsinization, triplicates of 4.8 × 103 cells were seeded on 48-well tissue culture plates, expanded in EM and fixed at d4 for 10 min in 4% FA and then stored at 4 °C in PBS. Beta galactosidase signals were detected with the CellEvent™ Senescence Green Detection Kit (ThermoFisher) according to the to the manufacturer’s protocol. In addition, nuclei staining using 1 µg/ml DAPI (Sigma-Aldrich) in dH2O was performed. Fluorescence imaging under constant settings was performed using a fluorescence microscope (Invitrogen EVOS FL). The mean beta galactosidase signal intensities of each image were quantified using ImageJ.
4
Senescence evaluation of melanoma cell lines
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A375, SKMEL-239, and M14 cells were cultured in a humidified atmosphere of 5% CO2 at 37°C in DMEM (Life Technologies, Carlsbad, CA, USA) or RPMI-1640 medium (Life Technologies), each supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). The percentage of senescent cells was measured using the Cell Event™ Senescence Green Detection Kit (Thermo Fisher Scientific, cat. no. C10850) according to the manufacturer’s instructions. The cells were seeded on 96-well plates at a density of 300 cells/well and allowed to adhere overnight. Then the cells were treated with vehicle or XL413 at concentrations of 1 μM and 2 μM for 7 days. The cells were then washed with PBS and fixed with 100 μL 2% paraformaldehyde solution per well for 10 min at room temperature. The cells were then washed with 100 μL 1% bovine serum albumin (BSA) in PBS, and 100 μL 1× working solution was added. The plate was incubated for 2 h at 37°C without CO2 in the dark. Then, the wells were washed three times with PBS to remove the working solution, 100 μL PBS/well was added, and 10× images (Scale: 200 μm) were captured using an Alexa FluorTM 488/fluorescein isothiocyanate (FITC) Filter Olympus Florescence microscope (IX73, Olympus, Tokyo, Japan).
5
Senescence Evaluation in HUVEC-MSC Coculture
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The HUVECs were cocultured with relative amounts of MSC‐mt as previously described. The inhibitors including U0126 (MedChem Express, #HY‐12031; 10 μM), or LY294002 (MedChem Express, #HY‐10108; 10 μM), or SB203580 (MedChem Express, #HY10256; 10 μM) were added and kept cultured for additional 24 h. Senescence‐associated b‐galactosidase (SA‐β‐gal) staining was performed according to the manufacturer's instructions (CellEvent Senescence Green Detection Kit, Thermo Fisher, #C10851 for flow cytometry analysis; SA‐β‐gal staining kit, Beyotime, #C0602 for cell staining).
6
Senescence-Associated β-Galactosidase Activity Assay
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SA-β-gal activity was tested using the CellEvent™Senescence Green Detection Kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s protocol. In brief, the cardiomyocytes were cultured on coverslips in a 6-well cell culture plate for 24 h. The cells were then fixed with 4% PFA for 0.5 h. After rinsing three times with PBS, the cells were treated with Sa-β-gal staining solution under CO2-free conditions. The cells were then treated with Hoechst 33342 (2 μg/mL) to stain cell nuclei for 10 min. Finally, the cell samples were checked by an FV3000 laser scanning confocal microscope (Olympus).
7
Senescence in B16-F10 Melanoma Cells
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B16-F10 cells were plated in 8-well chamber slides from ibidi (Gräfelfing, Germany) at a density of 20,000 cells/well. After 24 hours, cells were treated for two hours in serum-free media containing 7.5 µM docetaxel or 3.75 µM TH1902. Cells were then washed twice with complete growth medium and incubated for four days in complete growth medium. As a positive control for senescence, cells were treated for 24 hours in serum-free medium with 12.5 µM etoposide, washed, and incubated in complete growth medium for up to four days. CellEvent™ Senescence Green Detection Kit (Thermo Fisher, #C10850) was used to detect senescence-associated β-Galactosidase (SA-β-gal) activity according to manufacturer instructions. Photomicrographs were taken and digitalized at 20x magnification by confocal microscopy (Nikon A1plus, Melville, NY) then analyzed using NIH ImageJ Version 1.4.21 software. For cell morphologic assessment, cells were fixed with 10% formalin phosphate, colored with 0.5% crystal violet staining solution/20% methanol for 20 min at room temperature, washed in water, air dried, and observed by microscopy.
8
Senescence Detection by SA-beta-Galactosidase Assay
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SA-beta-galactosidase activity was detected by CellEvent™ Senescence Green Detection Kit (C10850, Thermo Fisher Scientific). Cells were fixed in 2% paraformaldehyde in PBS (diluted from 8% paraformaldehyde, RT 157–8, Electron Microscopy Sciences) for 10 min. The assay was carried out according to manufacturer’s instructions. Cells were stained with 10 μM Hoechst 33342 and 500 ng/ml Alexa Fluor™ 568 NHS Ester for 30 min, followed by two washings with PBS, then imaged by fluorescence microscopy at 10X magnification.
9
Senescence Detection in 3D Spheroids
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After the treatment (24 and 48 h), the spheroids were washed with PBS and fixated with a 2% paraformaldehyde solution for 10 min. The spheroids were washed in a 10% BSA solution in order to remove the fixation solution and then proceeded to stain the spheroids with the CellEvent™ Senescence Green Probe provided by the CellEvent™ Senescence Green Detection Kit (ThermoFisher Scientific, Waltham, MA USA) and were incubated for 2-and-a-half hours at 37 °C without CO2 and in the absence of light. After incubation, the spheroids were washed with PBS and the fluorescence was measured using λexc = 485 nm and λem = 535 nm with the Mithras LB 940 plate reader.
10
Senescence and Mitochondrial Assays
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Induction of senescence was measured at 48 h and 72 h after transfection using the CellEvent™ Senescence Green Detection Kit (Thermo Fisher Scientific). Assays were performed according to the manufacturer’s protocol and measured by flow cytometry. For mitochondrial depolarization and mass measurement, MitoViewTM 633 was used at a final concentration of 100 nM and measured by flow cytometry according to the manufacturer’s protocol.
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