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.
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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.
I would like to excise a large strand of DNA and insert a new one using CRISPR. My problem is that my strand will be a little over 1kb and I am not sure if this is going to be a limiting factor. Also, how long should the homology arms be for a region of this size?
Contamination can affect cell characteristics, i.e., growth, metabolism, and morphology leading to unreliable and erroneous experimental data. Depending on the source of contaminants, one can detect contamination by using a light microscope, gram stain, isothermal amplification, or PCR. Bacteria and fungi can usually be identified by optical microscopy. Mycoplasma in cell cultures cannot be detected visually. Hence, these microbes can go unnoticed for long periods and are determined using dedicated assays. Early and rapid identification of contaminants is vital to detect, handle and prevent contamination for good cell-culture practices. However, detection and identification can be challenging and tricky based on usual visual identifications. Hence it is essential to use a standard contamination detection kit to detect and maintain best practices.
Contamination can affect cell characteristics, i.e., growth, metabolism, and morphology leading to unreliable and erroneous experimental data. Depending on the source of contaminants, one can detect contamination by using a light microscope, gram stain, isothermal amplification, or PCR. Bacteria and fungi can usually be identified by optical microscopy. Mycoplasma in cell cultures cannot be detected visually. Hence, these microbes can go unnoticed for long periods and are determined using dedicated assays. Early and rapid identification of contaminants is vital to detect, handle and prevent contamination for good cell-culture practices. However, detection and identification can be challenging and tricky based on usual visual identifications. Hence it is essential to use a standard contamination detection kit to detect and maintain best practices.
Contamination can affect cell characteristics, i.e., growth, metabolism, and morphology leading to unreliable and erroneous experimental data. Depending on the source of contaminants, one can detect contamination by using a light microscope, gram stain, isothermal amplification, or PCR. Bacteria and fungi can usually be identified by optical microscopy. Mycoplasma in cell cultures cannot be detected visually. Hence, these microbes can go unnoticed for long periods and are determined using dedicated assays. Early and rapid identification of contaminants is vital to detect, handle and prevent contamination for good cell-culture practices. However, detection and identification can be challenging and tricky based on usual visual identifications. Hence it is essential to use a standard contamination detection kit to detect and maintain best practices.
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