Explore chapters and articles related to this topic
Cellular Injury Associated with Organ Cryopreservation: Chemical Toxicity and Cooling Injury
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
Gregory M. Fahy, Carla da Mouta, Latchezar Tsonev, Bijan S. Khirabadi, Patrick Mehl, Harold T. Meryman
Because apoptosis might not have a detectable effect on electrolyte transport, a similar experiment was carried out in which direct observation of DNA molecular weight distribution was used to check for the “ladders” characteristic of apoptosis in slices that had been previously cooled to −30°C and warmed to 25°C for 90 min. This preliminary experiment showed no obvious DNA “laddering” effect (data not shown).
Preclinical Characterization of Engineered Nanoparticles Intended for Cancer Therapeutics
Published in Mansoor M. Amiji, Nanotechnology for Cancer Therapy, 2006
Anil K. Patri, Marina A. Dobrovolskaia, Stephan T. Stern, Scott E. McNeil
Apoptosis in mammalian cells can be initiated by four potential pathways: (1) mitochondrial pathway, (2) Death receptor-mediated pathway, (3) ER-mediated pathway, and (4) Granzyme B-mediated pathway.114 Our laboratory has focused on caspase-3 activation in liver and kidney cells as a biomarker of apoptosis, since this a downstream event in all the classical apoptotic signaling pathways and can be measured using a fluorometric protease assay. This assay quantifies caspase-3 activation in vitro by measuring the cleavage of DEVD-7-amino-4-trifluoromethyl coumarin (AFC) to free AFC that emits a yellow-green fluorescence (λmax = 505 nm).115 This initial apoptosis screen can then be followed by additional analysis, as cellular morphology studies using nuclear staining techniques to detect perinuclear chromatin, or agarose gel electrophoresis to detect DNA laddering.116
Ovotoxic Environmental Chemicals: Indirect Endocrine Disruptors
Published in Rajesh K. Naz, Endocrine Disruptors, 2004
Patrick J. Devine, Patricia B. Hoyer
Apoptosis and necrosis can also be distinguished by certain biochemical features. Apoptosis often depends on ligand-receptor interactions (e.g., Fas/Fas ligand) and is an active, energy-requiring process. Triggering of apoptosis leads to altered localization of bcl-2 family members or activation of certain Caspases (Caspase 2, 3, 8, and 9), which in turn activate proteolytic and DNA-degrading enzymes.[18] Through studies involving gene-deficient mice, some genes such as Bax and Bcl-2 have been shown to influence follicle numbers by altering apoptosis.[19] In most cell types, genomic DNA is degraded in a specific internucleosomal pattern to produce low molecular weight fragments (180 base pairs) that appear as a characteristic “ladder” formation on agarose gels.[17] However, this pattern of DNA fragmentation is not observed in all cells undergoing apoptosis, including granulosa cells from immature (small pre-antral) ovarian follicles, in which DNA is degraded in a non-specific, random pattern.[20] This is because these cells do not express the specific endonuclease necessary to produce the normal pattern of DNA laddering in rats.[21] Thus, morphological evaluation remains the most reliable distinction between apoptotic and necrotic mechanisms of cell death.[22]
Anagallis arvensis Induces Apoptosis in HL-60 Cells Through ROS-Mediated Mitochondrial Pathway
Published in Nutrition and Cancer, 2021
Satyam Kumar Agrawal, Madhunika Agrawal, Parduman Raj Sharma, Khursheed Ahmad, Abdul Sami Shawl, Saroj Arora, Ajit Kumar Saxena
The method of DNA laddering was done as proposed by Sharma et al. (27) with slight modifications. In this method, HL-60 cells (1 × 106/ml) after treatment with different concentrations (5–20 µg/ml) of AAE for 6 h were centrifuged at 500g for 10 min, and washed with PBS. The resultant pellet was suspended in 250 μl of lysis buffer (10 mM EDTA, 50 mM Tris–HCl, 0.5% SDS) for 15 min at 55 °C. Lysed cells were then digested with proteinase-K (200 µg/ml) followed by incubation with 100 μg/ml DNase-free RNase A at 55 °C for 90 min. The DNA extraction was done with 250 µl of phenol: chloroform: isoamylalcohol (25:24:1) followed by centrifugation at 12,000g for 5 min twice. Chloroform: isoamylalcohol (24:1) was used to further extract the aqueous phase. DNA was precipitated from aqueous phase with 0.1 volume of 2 M NaCl and 2.5 volumes of chilled ethanol. This precipitate was centrifuged at 12,000g for 10 min. The DNA pellet was washed in 80% ethanol, dried, dissolved in 100 µl Tris–EDTA buffer (pH 8.0) and electrophoresed in 1.5% agarose gel at 50 V for 1.5 h. The gel was photographed by using Bio-Rad gel documentation system.
Polybia occidentalis and Polybia fastidiosa venom: a cytogenotoxic approach of effects on human and vegetal cells
Published in Drug and Chemical Toxicology, 2021
Marcel José Palmieri, Amanda Ribeiro Barroso, Larissa Fonseca Andrade-Vieira, Marta Chagas Monteiro, Andreimar Martins Soares, Pedro Henrique Souza Cesar, Mariana Aparecida Braga, Marcus Vinicius Cardoso Trento, Silvana Marcussi, Lisete Chamma Davide
Both venoms tested in this work induced an expressive amount of CNs (32.50% and 18.67% of the total CCAs for P. occidentalis and P. fastidiosa, respectively) (Figure 1(D) and 2). Also, 12.6% of the cells exposed to P. occidentalis and 10.6% of the cells exposed to P. fastidiosa venom were positive for the TUNEL signal (Table 1), which indicates DNA damage and fragmentation. The occurrence of DNA laddering was also visible on the agarose gel electrophoresis (Figure 3). As discussed before, mastoporans, hyaluronidases, and phospholipases can all disrupt a series of cell structures and membranes, and result in cell lyses and CD.
Isocyanate induces cytotoxicity via activation of phosphorylated alpha synuclein protein, nitrosative stress, and apoptotic pathway in Parkinson’s Disease model-SHSY-5Y cells
Published in Neurological Research, 2023
We used agarose and ethidium bromide gel electrophoresis to examine DNA laddering using the Kasibhatla et al. [21] protocol to examine apoptosis in SHSY-5Y cells after NSMC exposure. During apoptosis, endonuclease cleaves DNA into 180 base-pair oligomers, which can be visualized as a DNA ladder on agarose gels. Cells were trypsinized and collected for DNA extraction by phenol: chloroform-isoamyl alcohol extraction after incubation with NSMC for various time periods and concentrations. Afterward, the DNA samples were run on a 1.5% agarose gel and stained with ethidium bromide. Quantification of DNA was done by taking the absorbance at A260 nm. The purity was determined using the A260/A280 ratio (FLUOstar® Omega Plate Reader, BMG LABTECH). The concentration of DNA was calculated using the formula: Concentration (µg/ml) = A260 reading × dilution factor × 50 µg/ml. A 2% Agarose gel was prepared in 1× Tris-acetate EDTA (TAE) buffer by heating in the microwave. The solution was allowed to cool to about 55–60°C by swirling the flask occasionally to cool evenly, and 0.2 μg/mL ethidium bromide (EtBr) was added. Melted agarose solution was poured into the gel-casting tray fitted with combs and allowed to solidify. The gel was placed in the electrophoretic chamber and submerged in a sufficient amount of TAE buffer. In 1 μl of loading dye, 5 μl of the sample was mixed and loaded into the respective wells along with 0.6 μl of each low molecular weight marker (100bp) and high molecular weight marker (1Kb) mixed with 1 μl of loading dye. The electrophoresis instrument was connected to the power pack and allowed to run at 120 V for approximately 25–30 min. The bands formed in the gel were viewed under a UV–Visible Transilluminator, and a photograph was taken with the Gel Documentation System of Vilber Lourmat, France. The density of fragmented DNA was calculated using ImageLab software.