siRNA / miRNA gene silencing Human Min-6

Get tips on using Vybrant™ Apoptosis Assay Kit #4, YO-PRO™-1/Propidium Iodide to perform Apoptosis assay cell type - MG-63

Cell cytotoxicity assays measure the ability of certain compounds or chemical mediators to reduce the viability of the cells. The term cell cytotoxicity assay can sometimes be used interchangeably with cell proliferation assay. Healthy living cells can be identified by the use of formazan dyes, protease biomarkers or by measuring ATP content. The formazan dyes are chromogenic products formed by the reduction of tetrazolium salts by dehydrogenases, such as lactate dehydrogenase (LDH) and reductases that are released during cell death. Common tetrazolium salts include INT, MTT, MTS and XTT. Cell cytotoxicity can also be measured by using the SRB and WST-1 assays. These assays can usually be used in a high-throughput fashion and can be quantitated by measuring absorbance, colorimetry or luminescence. All these assays require similar numbers of cell plating at the initiation, a time course of treatment with the cytotoxic agent and at least triplicates for each condition at every point of analysis. Cell shrinkage, plasma membrane blebbing, cell detachment, externalization of phosphatidylserine, nuclear condensation and ultimately DNA fragmentation are well-described features of apoptosis. The assays that rely on cell membrane integrity for their function, may not be able to quantify early apoptosis. Therefore, in order to distinguish early apoptotic vs. late apoptotic or necrotic cells, additional flow cytometry techniques can be used. A combination of Annexin V and PI (propidium iodide) can be used to distinguish early (Annexin V+/PI-) and late apoptotic (Annexin V+/PI+) cells. Sometimes, caspase assays are used in order to differentiate the stages of apoptosis.

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 Gentra Puregene Blood Kit to perform DNA isolation / purification Cells - Primary cells Bone marrow mononuclear cells

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. The resulting amplicons are generally detected by gel electrophoresis and for some further applications like cloning, sequencing, amplicon product needs to be recovered from the gel and subsequently purified. However, non-specific product amplification and primer-dimer formation during set-up make gel extraction difficult. Nevertheless, high-quality DNA polymerase and optimize reaction buffers will certainly lead to a successful PCR reaction.

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. The resulting amplicons are generally detected by gel electrophoresis and for some further applications like cloning, sequencing, amplicon product needs to be recovered from the gel and subsequently purified. However, non-specific product amplification and primer-dimer formation during set-up make gel extraction difficult. Nevertheless, high-quality DNA polymerase and optimize reaction buffers will certainly lead to a successful PCR reaction.

Get tips on using TurboCapture 96 mRNA Kit (5) to perform mRNA / Ribonucleoprotein isolation / purification mRNA

Get tips on using TurboCapture 384 mRNA Kit (5) to perform mRNA / Ribonucleoprotein isolation / purification mRNA