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Get tips on using MammoCult™ Human Medium Kit to perform 3D Cell Culture Media Human breast cancer MCF-7 cells-Mammospheres

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.

ROS has a very short half-lives in biological environment as they are influenced by exposure to ambient oxygen. As it is highly reactive and hard to measure care should be taken to ensure the stability of the sample during isolation, preparation, storage, and analysis.

Get tips on using EpiQuik Dnmt3A Assay Kit to perform DNA methylation profiling Whole genome profiling - MCF-7, MDA-MB-453 human breast cancer

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.

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.

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.

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.

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.

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.