shRNA gene silencing Human Neuroblastoma cells (SH-SY5Y)

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.

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary human dermal fibroblasts

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary human osteoblasts - osteoarthritis

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary human lung fibroblasts

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary human epidermal melanocytes

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary human epidermal keratinocytes

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary human chondrocytes - osteoarthritis

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary human cardiac fibroblasts

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - immortalized Human blood cord

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.