Cellular senescence was determined by β-gal staining as described previously (Gey and Seeger, 2013 (link)). Apoptosis/necrosis was detected using the commercial kit (AB176749; Abcam, Cambridge, England) following the manufacturer’s instructions. The Apoptosis/Necrosis Detection Kit is designed to simultaneously monitor apoptotic, necrotic, and healthy cells with a flow cytometer or fluorescence microscope. In apoptotic cells, PS is transferred to the outer side of the plasma membrane and can be detected by its sensor, Apopxin Green (green fluorescence). The 7-aminoactinomycin D (red fluorescence), a membrane impermeable dye, will label the nucleus of damaged cells, including both late apoptotic and necrotic cells. Live cells can be detected as blue fluorescence by CytoCalcein Violet 450 staining. Cytotoxicity was also examined by propidium iodide permeability assay. After damage induction and inhibitor treatment, propidium iodide and Hoechst 33342 were added into the medium to achieve final concentrations of 0.8 and 0.5 µg/ml, respectively. The cells were then incubated at 37°C for 5–15 min and visualized for the total cells (blue, Hoechst 33342) and dead cells (red, propidium iodide) under a fluorescence microscope.
Ab176749
Manufactured by Abcam
Sourced in United Kingdom
Ab176749 is a recombinant monoclonal antibody. It is a research-use only product designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6, by BD (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Costar 3603, by Corning (1 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Middlebrook 7h11 agar plates, by BD (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Jc 1 mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
96 well flat clear bottom black microplates, by Corning (1 mentions)
O vanillin, by Merck Group (1 mentions)
Its 1x, by Thermo Fisher Scientific (1 mentions)
Rg7112, by Selleck Chemicals (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
13 protocols using ab176749
1
Cellular Senescence and Apoptosis Detection
2
Quantifying Apoptosis and Necrosis by Flow Cytometry
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An apoptosis/ necrosis assay (ab176749, Abcam, Cambridge, UK) was performed using flow cytometry to quantify apoptotic and necrotic cells. Cells were first detached, and cell pellets were washed with PBS. They were incubated for 1 h with the stain in the absence of light before conducting flow cytometry analysis. Apoptosis was quantified using a phosphatidylserine sensor which emits green fluorescence upon binding to membrane phosphatidylserine. Necrosis was quantified using 7-AAD, a red membrane-impermeable dye labelling the nucleus, demonstrating late-stage apoptosis or early necrosis. Flow cytometry measurements were performed using a BD Accuri™ C6 personal flow cytometer (BD Biosciences, USA) with 20,000 cells per sample for analysis. The FL1 channel was used to quantify apoptosis (PS sensor, Ex/Em = 490/525 nm), and the FL2 channel was used to quantify necrosis (7-AAD, Ex/Em = 546/647 nm).
3
Detecting Cell Death Mechanisms via Hyperthermia
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To further determine the type of cell death induced by HT, Capan-2 and BxPC-3 cells were labeled with an apoptosis/necrosis detection kit (ab176749, Abcam, Cambridge, UK). Cells were stained according to the manufacturers’ protocol immediately after removal from the hyperthermic conditions. This kit enables discrimination between healthy, apoptotic, and necrotic cells. Cells incubated at 55 °C for 1 h were used as a positive control for necrosis. Apoptosis was calculated relative to the respective cell lines growing at 37 °C, during the logarithmic growth phase. Images were acquired via Nikon Eclipse TI inverted microscope at a magnification of 4x. Quantitative analysis was performed using the Fiji/ImageJ software [42 (link)]. Microscopic evaluation was performed in sextuplicate.
4
Apoptosis and Necrosis Detection in Cells
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Cells (1 × 103 cells/well) were grown in 96-well plates with a clear flat bottom (Corning™ Costar™ 3603, USA) at 37 °C for 24 h. They were then treated by EPA at 100 μM or 200 μM, free fatty acid extract from krill oil at 0.12 μL/100 μM well for 48 h. After removing the media, cells were washed twice by using PBS. Apoptosis/Necrosis was detected using the commercial kit (ab176749, Abcam, Cambridge, England). Cells were examined under the fluorescence microscope (Olympus 1 × 81). The apoptosis was determined by comparing the number of treated cells against the control. Each experiment was performed in quadruplicates, and it was carried out three times for each cell line.
5
Prednisolone-Induced Apoptosis Assay
6
Detecting Apoptosis in FSHD Myocytes
7
Apoptosis Measurement by Flow Cytometry
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Apoptosis was detected by flow cytometry using the apoptosis assay kit (ab-176749 Abcam) as we have previously described [12 ].
8
Macrophage Response to M. tuberculosis
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Macrophages were lysed with 0.05% SDS at different time points post-M. tuberculosis infection with or without hGM-CSF supplementation, α-hGM-CSF antibodies, or other pharmacological agents/inhibitors. Cell culture media along with hGM-CSF and α-hGM-CSF antibodies were replaced every alternate day during these experiments and assays. At different time points, lysates were plated at serial 10-fold dilutions in PBS using 7H11 Middlebrook agar plates (Difco Laboratories, Surrey, UK). The plates were incubated at 37°C for 3 weeks before counting CFUs. Data were expressed as log10- CFUs per million macrophages. Alamar Blue cell viability reagent (Life Technologies, DAL1025) was used to assess cell viability by adding the ready-to-use 1X solution to uninfected or M. tuberculosis-infected macrophages at various time points, followed by fluorescence measurement through fluorimeter per the manufacturer’s protocol. A fluorescence-based apoptosis/necrosis detection kit (Abcam #ab176749) that can simultaneously monitor apoptotic, necrotic, and healthy cells was used to measure and quantify apoptosis and necrosis in M. tuberculosis infected, uninfected, differentiated, and undifferentiated macrophages using a fluorescence microscope.
9
Multiparametric Cell Death Assay
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An apoptosis/necrosis detection kit (ab176749, Abcam, MA, USA) was used to simultaneously monitor apoptotic (green), necrotic (red), and healthy (blue) cells in strict accordance with the manufacturer’s instructions. The stained samples were analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA) and fluorescence microscopy (LSM800, Carl Zeiss, Germany).
The MMP was measured using a JC-1 mitochondrial membrane potential assay kit (C2006, Beyotime Biotechnology, Shanghai, China). Cells were incubated with JC-1 solution at 37 °C for 20 min, and imaging was then performed using a high-content imaging system (Harmony, PerkinElmer, Rodgau, Germany). The ratio of red to green fluorescence intensity was used to determine the MMP.
The MMP was measured using a JC-1 mitochondrial membrane potential assay kit (C2006, Beyotime Biotechnology, Shanghai, China). Cells were incubated with JC-1 solution at 37 °C for 20 min, and imaging was then performed using a high-content imaging system (Harmony, PerkinElmer, Rodgau, Germany). The ratio of red to green fluorescence intensity was used to determine the MMP.
10
PAuNPs Enhance X-ray Sensitivity in Cancer Cells
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