Thermo Scientific Cellomics Multi parameter cytotoxicity 3 Kit (Thermo Scientific, Japan) was used to enable simultaneous measurement of cell permeability, cell count, nuclear intensity, mitochondrial membrane potential, and cytochrome c level for the MCF7 cell line. The kit used contains cytochrome c (primary antibody), DyLight™ 649 conjugated goat anti-mouse IgG. It also contains mitochondrial membrane potential, permeability and Hoechst dyes along thin plate seal assembly. Wash buffer (10X Dulbecco’s PBS), permeabilization buffer (10X Dulbecco’s PBS with 1% Triton® X-100), and blocking buffer (10X) were used. The kit enables measurements of several cell parameters at the same time. The distribution and intensity of fluorescence within cell line (N=5) was imaged with HCS system (Thermo Scientific). The HCS system was attached to a computerized imaging microscope equipped with Zeiss 40X (0.75 NA) Plan-Neofluar objective lens. The cells were treated for anastrozole for 24 hours followed by the addition of Mitochondrial membrane potential (MMP) and the cell permeability dyes and then incubated 37°C for 30 min. The cells were fixed and permeabilized using a standard procedures.23
Hcs system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HCS) system is a high-content screening platform developed by Thermo Fisher Scientific. The system provides automated image acquisition and analysis capabilities for cellular-based assays. It enables researchers to study cellular responses to various stimuli at the single-cell level.
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5 protocols using hcs system
1
Multiparametric Cytotoxicity Assay for MCF7 Cells
2
Measuring Epithelial-Mesenchymal Transition in HepG2 Cells
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HepG2 human liver cancer cells (2 × 105 cells/mL) were seeded into 24-well plates. The cells were divided into six groups as described in the wound-healing assay. After the incubation for 24 h, cells were washed twice in PBS, fixed in 10% cold formalin (−20°C), and then blocked with FBS (5% BSA and 0.1%Tritonx-100) for 1 h. The cells were then incubated in the same solution containing primary antibodies specific for E-cadherin (1:100 dilution) and vimentin (1:100 dilution) for 1 h at room temperature. After three washes with phosphate-buffered saline (PBS), the cells were incubated in a secondary antibody (1:200 dilution) for 30 min at room temperature, incubated with DAPI for 10 min at room temperature, washed twice with PBS, and then observed using a high-content screening (HCS) system (Thermo Fisher, USA).
3
Fluorescence-based ROS Measurement
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Measurement of ROS was performed as previously described (Peng et al., 2021 (link)). The DHE probes were diluted 1:1,000 with serum-free medium to a final concentration of 10 μM. The serum-free cell culture medium was removed, and an appropriate volume of diluted DHE was added. The appropriate volume to cover the cells was added and the cells were incubated for 20 min in a 37°C cell incubator. The cells were washed three times with serum-free cell culture to adequately remove DHE that did not enter the cells. The high-content screening (HCS) system (Thermo, Waltham, MA, USA) was used to take fluorescence images and analyze the data (excitation wavelengths = 549 nm).
4
Quantitative Immunofluorescence Assay
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The transfected cells were plated into 96-well plates at a density of 4000 cells/well. The cells were fixed with ice-cold methanol and blocked with serum. The primary antibodies of VEGFR1, VEGFR2, and VE–Cad (Abcam, USA) were used at a 1:100 work solution. Fluorescein isothiocyanate- and tetramethyl rhodamine isothiocyanate-conjugated mouse and rabbit IgG antibodies (Santa Cruz Biotechnology, USA) were used to label immunofluorescence. After immunolabeling, the cells were stained with Hoechst (Sigma, Germany) and measured with high-content screening (HCS) systems to evaluate the protein expression (Thermo Scientific, USA).
5
Apigenin's Impact on Cell-Cell Adhesion Proteins
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Bel-7402 and PLC cells (4 × 104 cells/mL) were seeded on 96-well culture plates. After overnight incubation, the cells were treated with various concentrations of apigenin. After incubation for 24 h, the cells were washed twice in PBS, fixed with 10% formalin in PBS, permeabilized, and blocked with PBS containing NP-40 (0.1%) and BSA (3%). The cells were then incubated in the same solution containing primary antibodies specific for E-cadherin (1:50 dilution), claudin3 (1:50 dilution), vimentin (1:50 dilution), or N-cadherin (1:50 dilution) for 1 h at room temperature (25°C). The cells were washed four times in PBS and incubated in secondary antibody (1:200 dilution) for 30 min at room temperature (25°C). The cells were then washed four times in PBS and covered with Hoechst 33342 dye for 30 min at room temperature. The cells were again washed four additional times in PBS; thereafter, proteins were visualized with high-content screening (HCS) systems (Thermo Fisher, USA).
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