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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.

Cellular assays Autophagy assay cell type Mouse aortic endothelial cells (MAECs)

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.

RNA RNA isolation / purification Cells primary human islets of langerhans

Get tips on using CelLytic™ MT Cell Lysis Reagent to perform Protein isolation Mammalian cells - Human CD14+ cells

Products Sigma-Aldrich CelLytic™ MT Cell Lysis Reagent

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary mouse tracheal epithelial cells

Products Thermo Fisher Scientific TRIzol Reagent

Get tips on using NucleoSpin® RNA to perform RNA isolation / purification Cells - primary mouse cortical neurons

Products Macherey Nagel NucleoSpin® RNA

Get tips on using RNeasy Micro Kit to perform RNA isolation / purification Cells - primary mouse cardiac fibroblasts

Products Qiagen RNeasy Micro Kit

Get tips on using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents to perform Protein isolation Mammalian cells - Rat_Liver

Products Thermo Fisher Scientific NE-PER™ Nuclear and Cytoplasmic Extraction Reagents

Get tips on using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents to perform Protein isolation Mammalian cells - HEK293T

Products Thermo Fisher Scientific NE-PER™ Nuclear and Cytoplasmic Extraction Reagents

Get tips on using RNeasy Plus Mini Kit to perform RNA isolation / purification Cells - primary mouse oocytes

Products Qiagen RNeasy Plus Mini Kit

Get tips on using Mouse MPO/Myeloperoxidase PicoKine™ ELISA Kit Skip to the end of the images gallery to perform ELISA Mouse - MPO

Products BosterBio Mouse MPO/Myeloperoxidase PicoKine™ ELISA Kit Skip to the end of the images gallery

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