rna-isolation-purification-cells-primary-rat-brain-microvascular-endothelial-cells

TUNEL assay is the cell death detection method where the biochemical marker of apoptosis is DNA fragmentation. The assay involves the microscopical detection of generated DNA fragments with free 3'-hydroxyl residues. in apoptotic cells using enzyme terminal deoxynucleotidyl transferase (TdT) which adds biotinylated nucleotides at the site of DNA breaks. Major challenges of this method involve proper access of the enzyme which could be hampered by poor permeabilization and/or excessive fixation with cross-linking fixative (common with archival tissue). This issue can be resolved by optimizing the incubation time with Proteinase K or CytoninTM.

ELISA is the most commonly used method of detecting and quantifying the concentration of an antigen in an unknown sample. During the experiment, If you get a weak signal, then make sure reagents are at room temperature before starting the assay. Try increasing incubation times to ensure maximal antibody binding and amplify the signal. Secondly, if you get values above 0 in the negative control indicates a high background signal. Try to consider reducing your antibody concentration and prevent non-specific binding of antibodies by using affinity-purified antibody and suitable blocking buffers. To avoid high well to well variation, do not stack plates during incubation, no bubbles in the plate and wash wells thoroughly to avoid variation.

Get tips on using Slc1a2 to perform siRNA / miRNA gene silencing Rat - Glial cells GLT-1

Get tips on using LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells to perform Live / Dead assay mammalian cells - mouse bone marrow-derived macrophages

Get tips on using Gabpb1 siRNA to perform siRNA / miRNA gene silencing Rat - Schwann cells Nrf2

Get tips on using Ccr2 siRNA to perform siRNA / miRNA gene silencing Rat - Glial cells CCR2

Get tips on using ExpressArt FFPE Clear RNAready to perform RNA isolation / purification Tissue - human kidney tissue

Get tips on using RNAprep pure Kit (For Plant) to perform RNA isolation / purification Yeast - Neurospora crassa

Get tips on using Qubit RNA HS Assay Kit to perform RNA quantification Fuorimetric - human trophoblast cells

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.