As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.
As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.
As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.
Get tips on using Xfect™ Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Human pulmonary artery smooth muscle cells (HPASMC)
Get tips on using BrdU Cell Proliferation Assay Kit to perform Cell cytotoxicity / Proliferation assay cell type - BxPc-3 human primary pancreatic adenocarcinoma
Get tips on using MEBMTM Mammary Epithelial Cell Growth Basal Medium to perform 3D Cell Culture Media Human primary breast ephitelial cells-Mammospheres
Get tips on using DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit to perform ROS assay cell type - human primary corneal epithelial cells
DNA isolation and purification is the first critical step in sample preparation that helps ensure optimal performance of downstream assays like PCR, microarrays, and sequencing. Failure in yielding high-quality DNA would be the major reason that DNA doesn't work for the downstream application. To circumvent this, one should follow the recommended storage conditions to minimize DNA degradation by nucleases and shouldn't overload the purification system.
The estimation of DNA methylation level heavily depends on the complete conversion of non-methylated DNA cytosines. It is crucial to ensure complete conversion of non-methylated cytosines in DNA. Therefore, it is important to incorporate controls for bisulfite reactions, as well as to pay attention to the appearance of cytosines in non-CpG sites after sequencing, which is an indicator of incomplete conversion.
The estimation of DNA methylation level heavily depends on the complete conversion of non-methylated DNA cytosines. It is crucial to ensure complete conversion of non-methylated cytosines in DNA. Therefore, it is important to incorporate controls for bisulfite reactions, as well as to pay attention to the appearance of cytosines in non-CpG sites after sequencing, which is an indicator of incomplete conversion.
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