DNeasy Blood & Tissue Kit (69504, QIAGEN) was used to extract genomic DNA from liver tissues. A mitochondrial DNA isolation kit (ab65321, abcam) was employed to prepare the mitochondrial DNA from liver tissues. The following primer sequences were used to amplify nuclear and mitochondrial DNA sequences: PKLR 5′-CCAGCAGCATCAGTCGTATATC, PKLR 3′-ACCCAGGAGGAATC GAATTAAC; ND6 5′-GTTTGGGAGATTGGTTGATGTATG, ND6 3′-CACCCAGCTACTACCATCATTC.
Ab65321
Manufactured by Abcam
Ab65321 is a laboratory equipment product. It is designed for a specific function within a laboratory setting. No further details about its core function or intended use can be provided in an unbiased and factual manner.
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5 protocols using ab65321
1
Liver Tissue DNA Extraction and Amplification
2
Bone Marrow-Derived Macrophage Activation
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BMDMs were generated as previously described (Zhou et al., 2018 ). In brief, bone marrow cells were isolated from femurs and tibias of young and aged mice. The cells were cultured in DMEM supplemented with 10% fetal bovine serum and 20% L929‐conditioned medium for 7 days. The BMDMs were replated and cultured overnight for further experiments.
BMDM stimulation and activation studies: the hepatocytes were subjected to the HR model for 12 hr, the hepatocytes and supernatant were collected, and the mtDNA was extracted from the HR‐stressed hepatocytes using a mitochondrial DNA isolation kit following the instructions (ab65321; Abcam). After incubation with the above hepatocytes (BMDM/hepatocyte at a ratio of 2:1), supernatant or mtDNA (100 ng/ml) for 6 hr, the BMDMs and supernatant were harvested for further analysis.
BMDM stimulation and activation studies: the hepatocytes were subjected to the HR model for 12 hr, the hepatocytes and supernatant were collected, and the mtDNA was extracted from the HR‐stressed hepatocytes using a mitochondrial DNA isolation kit following the instructions (ab65321; Abcam). After incubation with the above hepatocytes (BMDM/hepatocyte at a ratio of 2:1), supernatant or mtDNA (100 ng/ml) for 6 hr, the BMDMs and supernatant were harvested for further analysis.
3
Mitochondrial DNA Extraction and Analysis
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About 0.5 × 107 senescent or non-senescent cells were treated with 0.5 μM SSK1 or DMSO in this assay. After treatment, the cells were washed with PBS and collected for mitochondrial DNA extraction by using the kit (Abcam, ab65321). The concentration of mitochondrial DNA was measured by nanodrop. Then 500 ng mitochondrial DNA from each sample was used and separated by 1% agarose gel for analysis.
4
Mitochondrial DNA Sequencing Protocol
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Oocytes were collected and used for mtDNA isolation following manufacturer's guidelines (ab65321; Abcam). Harvested mtDNA were detected by the agarose gel electrophoresis and quantified by QubitR 2.0 fluorometer (Thermo Fisher). Sequencing libraries were generated using a NEBNextR Ultra DNA library Pre Kit from Illumina (NEB, USA) following manufacture's protocol, and index codes were added to each sample. Libraries were analyzed for size distribution by Agilent2100 Bioanalyzer and quantified by qPCR. The libraries were sequenced using Illumina PE150. Trimmomatic‐0.38 was used for data filter and data cleaning. Bwa‐0.7.12 was employed for data mapping to the reference sequence (accession NC_0 05089.1), and GATK‐3.8‐0, Samtools‐1.9 and Varscan‐v2.4.3 were used for SNP/Indel detection.[38 (link)
] The mutations were not included if their minimal heteroplasmic fraction was less than 1% per strand.
] The mutations were not included if their minimal heteroplasmic fraction was less than 1% per strand.
5
Liver Tissue DNA Extraction and Amplification
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DNeasy Blood & Tissue Kit (69504, QIAGEN) was used to extract genomic DNA from liver tissues. A mitochondrial DNA isolation kit (ab65321, abcam) was employed to prepare the mitochondrial DNA from liver tissues. The following primer sequences were used to amplify nuclear and mitochondrial DNA sequences: PKLR 5′-CCAGCAGCATCAGTCGTATATC, PKLR 3′-ACCCAGGAGGAATC GAATTAAC; ND6 5′-GTTTGGGAGATTGGTTGATGTATG, ND6 3′-CACCCAGCTACTACCATCATTC.
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