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.
When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.
Get tips on using CelLytic™ MT Cell Lysis Reagent to perform Protein isolation Mammalian cells - Human CD14+ cells
Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary mouse tracheal epithelial cells
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Get tips on using RNeasy Micro Kit to perform RNA isolation / purification Cells - primary mouse cardiac fibroblasts
Get tips on using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents to perform Protein isolation Mammalian cells - Rat_Liver
Get tips on using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents to perform Protein isolation Mammalian cells - HEK293T
Get tips on using RNeasy Plus Mini Kit to perform RNA isolation / purification Cells - primary mouse oocytes
Get tips on using Mouse MPO/Myeloperoxidase PicoKine™ ELISA Kit Skip to the end of the images gallery to perform ELISA Mouse - MPO
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