DNA degradation was investigated in heart tissue, after extraction of DNA, using a specific Apoptotic DNA Ladder Kit (Roche Diagnostics GmbH, Mannheim, Germany), and following the specific procedures provided by the kit. The extracted DNA was loaded in a 1.7% agarose gel.
Apoptotic dna ladder kit
Manufactured by Roche
Sourced in Germany, Switzerland, United States
The Apoptotic DNA Ladder Kit is a laboratory product designed to detect and analyze DNA fragmentation, a hallmark of apoptosis or programmed cell death. The kit provides the necessary reagents and protocols to isolate and visualize the characteristic DNA ladder pattern associated with apoptotic cells.
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Lab products found in correlation
Cell death detection elisaplus kit, by Roche (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Diisopropylfluorophosphate, by Merck Group (1 mentions)
Rpmi 1640 cell medium, by Thermo Fisher Scientific (1 mentions)
2 4 dinitrophenylhydrazine, by Merck Group (1 mentions)
Enzchek caspase 3 assay kit, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Reduced glutathione, by Merck Group (1 mentions)
Dna molecular weight marker xiv, by Roche (1 mentions)
Vitamin d3, by Merck Group (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Mtt assay kit, by American Type Culture Collection (1 mentions)
Caspase 3 assay kit, by Beyotime (1 mentions)
Trypan blue solution, by Thermo Fisher Scientific (1 mentions)
Cell culture media, by Thermo Fisher Scientific (1 mentions)
Cleaved parp and cleaved caspase3, by Cell Signaling Technology (1 mentions)
Serum supreme, by Lonza (1 mentions)
32 protocols using apoptotic dna ladder kit
1
DNA Degradation Analysis in Heart Tissue
2
Optimized Apoptosis and Viability Assays
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Reduced glutathione (GSH), digitonin, dimethyl sulfoxide (DMSO), 2,2,2-trichloroacetic acid (TCA), 2,4-dinitrophenylhydrazine (DNPH), 5,5’-dithiobionitrobenzoic acid (DTNB) and diisopropyl fluorophosphate (DFP) were purchased from Sigma Chemical Company (St. Louis, MO). DMEM, KSFM and growth factors, and RPMI 1640 cell medium, penicillin/streptomycin solution, and geneticin (G418) and KB plus DNA ladder, Celltracker blue (7-amino-4-chloromethylcoumarin or CMAC), 10kD spin columns, and EnzChek Caspase-3 assay kit were purchased from Invitrogen (Grand Island, NY). BCA kit and the anti-DYKDDDDK (anti-FLAG) antibody (PA1-984B) were purchased from Pierce (Rockford, IL). Celltiter 96 AQueous One MTS kit, described as the MTS viability assay in experiments, was purchased from Promega (Madison, WI) and contained CellTiter96 Aqueous One Solution composed of a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium, inner salt (MTS) and an electron coupling reagent (phenazine methosulfate). The Apoptotic DNA ladder kit was purchased from Roche (Indianapolis, IN). All chemicals used for the synthesis of prodrugs were purchased from Sigma-Aldrich (St. Louis, MO), TCI (Portland, OR), Acros Organics (Thermo Fisher Scientific, New Jersey) and Lancaster (Ward Hill, MA) and used without further purification.
3
Apoptotic DNA Ladder Assay for CTOS
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Four hundred CTOS were treated with 1 mg/mL or 0.01 mg/mL EPI or MMC. The CTOS exposed to 1 mg/mL EPI or MMC were washed after 2 h exposure, and incubated in StemPro hESC. Three days after treatment, CTOS were collected and analyzed to detect DNA laddering using the Apoptotic DNA Ladder Kit (Roche, Mannheim, Germany) according to the manufacturer's protocol. Briefly, the collected CTOS were lysed and the DNA was extracted. The extracted DNA was electrophoresed on a 1% agarose gel. The gel was immersed in 10 mg/mL ethidium bromide solution for 15 min. The electrophoresed DNA was visualized under UV light.
4
Allicin-Induced DNA Fragmentation in Leishmania
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Qualitative analysis of DNA fragmentation was performed by agarose gel electrophoresis of total genomic DNA extracted from untreated and allicin treated promastigotes. Leishmania promastigotes were exposed to allicin (30 μM and 60 μM) for 24, 48 and 72h. After drug exposure cells were washed twice in sterile PBS (1000 xg, 10 min, RT) and the total DNA was extracted from the cell pellet (108 promastigotes) using the Apoptotic DNA ladder kit (Roche) following manufacturer’s protocol. The DNA was quantified at 260/280 nm using a NanoDrop ND-1000 spectrophotometer. The genomic DNA (5 μg) was run on a 2% agarose gel containing SYBR Safe DNA gel stain for 1 h at 100 V and visualized under UV light using the DNA Molecular Weight marker XIV (Roche).
5
Evaluating Arsenic Trioxide and Vitamin D3 Effects
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Arsenic trioxide, Lot No. 091419, CASRN 1327-53-3, MW 197.84, with an active ingredient of 100% (w/v) arsenic in 10% nitric acid was purchased from Fisher Scientific (Houston Texas). Vitamin D3, Lot No. 120M1635V was purchased from Sigma Aldrich (St. Louis, MO). Growth medium RPMI 1640 containing 1 mmol/L L-glutamine and fetal bovine serum (FBS) were purchased from Gibco BRL products (Grand Island, NY). Penicillin-Streptomycin, Lot No. 3000880, phosphate buffered saline (PBS-pH 7.4) Lot No. 3000892, and MTT assay kit Lot No. 3000800 were obtained from the American Type Culture Collection - ATCC (Manassas, VA). Annexin V-FITC Apoptosis Detection Kit Lot No. 42191 was purchased from BD Biosciences (San Diego, CA). Apoptotic DNA-Ladder kit, Lot No. 11324200 was purchased from Roche Molecular Biochemicals (Indianapolis, IN).
6
Measuring Cerebrovascular Apoptosis Markers
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Caspase-3 activity levels in isolated cerebral microvessels as well as murine cerebrovascular endothelial cells were assayed as previously described by Yin et al. with minor modifications17 (link). Briefly, 1 × lysis buffer was applied to lyse cerebrovascular endothelial cells. Following 10 min on ice, the cell lysate was spun down for 10 min at 10000 g (4 °C). The resulting supernatant was subjected to a caspase-3 activity assay according to the kit’s instructions (Caspase-3 Assay Kit, Beyotime Institute of Biotechnology). A microtiter plate reader was used to read optical density (OD) values at 405 nm. Relative caspase-3 activity was calculated by comparing OD levels from the experimental groups against those of controls.
DNA fragmentation levels in isolated cerebral microvessels were assayed as previously described by Yin et al.17 (link). Briefly, we first extracted the genomic DNA from isolated cerebral microvessels. Then, a commercial apoptotic DNA-ladder kit (Roche) was employed to measure DNA fragmentation levels.
DNA fragmentation levels in isolated cerebral microvessels were assayed as previously described by Yin et al.17 (link). Briefly, we first extracted the genomic DNA from isolated cerebral microvessels. Then, a commercial apoptotic DNA-ladder kit (Roche) was employed to measure DNA fragmentation levels.
7
Synthesis and Characterization of Ruthenium Complexes
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Ru(bathophenanthroline)3 (1 ) and Ru(bathophenanthroline disulfonate)3 (2 ) were synthesized using previously established
procedures.20 (link),44 All cell lines were purchased
from ATCC. Cell culture media, heat-inactivated
fetal bovine serum (FBS), 4–20% tris-glycine precast gels,
Dulbecco’s phosphate buffered saline (DPBS), penicillin-streptomycin
solution (pen-strep), and 0.4% Trypan Blue solution were from Invitrogen.
35 mm wide, 4-compartment CELLview cell culture dishes were obtained
from USA Scientific. Serum supreme was from Lonza. Hoechst 33342,
Lysotracker Green DND-26 and Mitotracker Green FM were purchased from
Invitrogen. Propidium iodide (PI) and FITC-Annexin V were obtained
from BD Science. Trimethylrhodamine ethyl ester (TMRE) was purchased
from Sigma–Aldrich. Antibodies for PARP-1, procaspase 3, and
GAPDH were from Santa Cruz Biotechnology, Inc., while cleaved PARP
and cleaved caspase 3 was from Cell Signaling Technology. RIPA buffer
was purchased from Santa Cruz Biotechnology, Inc. Clarity Western
ECL Substrate was from Bio-Rad. An apoptotic DNA-ladder kit was purchased
from Roche Applied Science.
procedures.20 (link),44 All cell lines were purchased
from ATCC. Cell culture media, heat-inactivated
fetal bovine serum (FBS), 4–20% tris-glycine precast gels,
Dulbecco’s phosphate buffered saline (DPBS), penicillin-streptomycin
solution (pen-strep), and 0.4% Trypan Blue solution were from Invitrogen.
35 mm wide, 4-compartment CELLview cell culture dishes were obtained
from USA Scientific. Serum supreme was from Lonza. Hoechst 33342,
Lysotracker Green DND-26 and Mitotracker Green FM were purchased from
Invitrogen. Propidium iodide (PI) and FITC-Annexin V were obtained
from BD Science. Trimethylrhodamine ethyl ester (TMRE) was purchased
from Sigma–Aldrich. Antibodies for PARP-1, procaspase 3, and
GAPDH were from Santa Cruz Biotechnology, Inc., while cleaved PARP
and cleaved caspase 3 was from Cell Signaling Technology. RIPA buffer
was purchased from Santa Cruz Biotechnology, Inc. Clarity Western
ECL Substrate was from Bio-Rad. An apoptotic DNA-ladder kit was purchased
from Roche Applied Science.
8
Apoptosis Pathway Activation Analysis
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Bortezomib (Velcade®) and antibodies against Bax, tubulin, cytochrome c, GAPDH, caspase-3, caspase-9, and PARP were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against BID and cleaved caspase-3 were purchased from Cell Signaling Technologies (Beverly, MA, USA). XIAP, cIAP1, and caspase-8 antibodies were purchased from R and D (USA). BD Cytofix/Cytoperm plus fixation and permeabilization solution kit with BD GolgiPlug, propidium iodide (PI) staining solution, annexin V binding buffer, mitochondrial membrane potential detection (JC-1) kit, stain buffer (FBS), annexin V-FITC antibody, H2AX (pS139)-Alexa Fluor 647 antibody, rabbit anti-active caspase-3- Bv605 antibody and PARP cleaved form-AF700 antibody were obtained from BD Biosciences (NJ, USA). Apoptotic DNA-ladder kit was obtained from Roche (Penzberg, Germany).
9
DNA Fragmentation Analysis via Apoptosis
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DNA in the cells after treatment was isolated using an Apoptotic DNA Ladder Kit (Roche, Indianapolis, IN). Briefly, cell suspension (200 μl) in PBS was mixed with 200 μl of binding buffer. After incubation for 10 min at room temperature, 100 μl of isopropanol was added to the sample and mixed by vortexing. DNA was then purified through the use of glass fibers. DNA samples were separated on a 2% agarose gel and visualized with ethidine bromide staining under UV light.
10
Apoptotic DNA Fragmentation Analysis
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HEMn were incubated with or without 500 μM SPER/NO for 24 h. Adherent and detached cells were then collected, and genomic DNA was extracted from the cells with the Apoptotic DNA Ladder Kit (Roche, Basel, Switzerland). The extracted DNA and the DNA ladder marker (Gene Ruler™ 1000 bp DNA Ladder, Thermo Scientific™) were loaded onto 1% agarose gel, and DNA fragments were separated by electrophoresis. The DNA ladder pattern was visualized on a UV transilluminator by SYBR®Green staining (Sigma-Aldrich, St. Louis, MO, USA). Images were obtained using a ChemiDOC™ XRS+Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).
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