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Can you suggest live cell proliferation assays at multiple time...
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Martin Bayer
Can you suggest live cell proliferation assays at multiple time...
I used CYQUANT (Molecular Probes) fluorescent based 96 well plate assay and aqueous one solution from Promega (optical density). Both worked well for me
I used CYQUANT (Molecular Probes) fluorescent based 96 well plate assay and aqueous one solution from Promega (optical density). Both worked well for me
Cell titer blue or alamar blue can be used for this. It's a non-toxic dye and travels across the cell membrane so no fixing or lysing is necessary, but you have to be sure you thoroughly rinse afterwards (several media changes should be fine) and use the same protocol, gain, etc.
Cell titer blue or alamar blue can be used for this. It's a non-toxic dye and travels across the cell membrane so no fixing or lysing is necessary, but you have to be sure you thoroughly rinse afterwards (several media changes should be fine) and use the same protocol, gain, etc.
To work without dye or other chemical, we use the x-celligence system that report in real time the proliferation of adherent cells. It works with plates lined with an electrode measuring electrical impedance that estimates the surface of the wells occupied by cells. http://www.aceabio.com/product.aspx You can then have proliferation curves for multiple wells in real time without any chemicals. Although you have to seed the cells directly on the plate (not on a coverslip), you might want to check if it is possible to place a matrix between the cells and the bottom of the well. Plates with "windows" are available: a part of the bottom's surface is devoid of electrode, this way you can monitor either cell morphology or staining of the cells (we do immunofluorescence).
To work without dye or other chemical, we use the x-celligence system that report in real time the proliferation of adherent cells. It works with plates lined with an electrode measuring electrical impedance that estimates the surface of the wells occupied by cells. http://www.aceabio.com/product.aspx You can then have proliferation curves for multiple wells in real time without any chemicals. Although you have to seed the cells directly on the plate (not on a coverslip), you might want to check if it is possible to place a matrix between the cells and the bottom of the well. Plates with "windows" are available: a part of the bottom's surface is devoid of electrode, this way you can monitor either cell morphology or staining of the cells (we do immunofluorescence).
Hi, Christopfer, if you have a good fluoresent microscope, you can try couple of fluoresent marker relate with cell proliferation. Fluoresent dyes, as Daniel mention, is also a good idea. But in some case dyes may have an effect on cell prolifaretion, if you incubate it for a long time. In ideal case you can create a tarcking system whcih can take images automatically once per 20-30 minutes, for example. If you have histone:GFP marler. you can easy quantifies cell divsion ratio, as well as cell cycle duration etc. But you need to have a precise automatical tracking system.. Good luck!
Hi, Christopfer, if you have a good fluoresent microscope, you can try couple of fluoresent marker relate with cell proliferation. Fluoresent dyes, as Daniel mention, is also a good idea. But in some case dyes may have an effect on cell prolifaretion, if you incubate it for a long time. In ideal case you can create a tarcking system whcih can take images automatically once per 20-30 minutes, for example. If you have histone:GFP marler. you can easy quantifies cell divsion ratio, as well as cell cycle duration etc. But you need to have a precise automatical tracking system.. Good luck!
If the latter case proves true, then you could probably adopt a high-throughput assay by lysis of your cells at different times, and quantifying total DNA by Cyquant on a plate reader. Would revert to end-point assays, but there may be no better option if your system is not compatible with microscopy.
If the latter case proves true, then you could probably adopt a high-throughput assay by lysis of your cells at different times, and quantifying total DNA by Cyquant on a plate reader. Would revert to end-point assays, but there may be no better option if your system is not compatible with microscopy.
Dear Christopher Charles West, BrdU, EdU, MTT, XTT, WST, Fluorescent Dye Proliferation Assays, trypan blue or Calcein-AM are popular Cell Proliferation Assay techniques. In my opinion, Rapid Colorimetric Assay might be a quick and simple assays method that would allow you to do this assessment at multiple time points in the same well. Cell Proliferation Proliferating cells incorporate the radioactive label into their nascent DNA, which can be washed, adhered to filters and then measured using a scintillation counter. Assays to measure cellular proliferation, cell viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. MTT Calorimetric Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or Calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells. For you question, to assess the growth at different time points e.g. 24,48,72 hours, and beyond, but without having to fix/freeze/kill the cells. It is advised to go ahead with Rapid Colorimetric Assay for cellular growth and survival. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. It can measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis. Ashish
Dear Christopher Charles West, BrdU, EdU, MTT, XTT, WST, Fluorescent Dye Proliferation Assays, trypan blue or Calcein-AM are popular Cell Proliferation Assay techniques. In my opinion, Rapid Colorimetric Assay might be a quick and simple assays method that would allow you to do this assessment at multiple time points in the same well. Cell Proliferation Proliferating cells incorporate the radioactive label into their nascent DNA, which can be washed, adhered to filters and then measured using a scintillation counter. Assays to measure cellular proliferation, cell viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. MTT Calorimetric Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or Calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells. For you question, to assess the growth at different time points e.g. 24,48,72 hours, and beyond, but without having to fix/freeze/kill the cells. It is advised to go ahead with Rapid Colorimetric Assay for cellular growth and survival. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. It can measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis. Ashish
Dear Christopher Charles West, BrdU, EdU, MTT, XTT, WST, Fluorescent Dye Proliferation Assays, trypan blue or Calcein-AM are popular Cell Proliferation Assay techniques. In my opinion, Rapid Colorimetric Assay might be a quick and simple assays method that would allow you to do this assessment at multiple time points in the same well. Cell Proliferation Proliferating cells incorporate the radioactive label into their nascent DNA, which can be washed, adhered to filters and then measured using a scintillation counter. Assays to measure cellular proliferation, cell viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. MTT Calorimetric Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or Calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells. For you question, to assess the growth at different time points e.g. 24,48,72 hours, and beyond, but without having to fix/freeze/kill the cells. It is advised to go ahead with Rapid Colorimetric Assay for cellular growth and survival. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. It can measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis. Ashish
Dear Christopher Charles West, BrdU, EdU, MTT, XTT, WST, Fluorescent Dye Proliferation Assays, trypan blue or Calcein-AM are popular Cell Proliferation Assay techniques. In my opinion, Rapid Colorimetric Assay might be a quick and simple assays method that would allow you to do this assessment at multiple time points in the same well. Cell Proliferation Proliferating cells incorporate the radioactive label into their nascent DNA, which can be washed, adhered to filters and then measured using a scintillation counter. Assays to measure cellular proliferation, cell viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. MTT Calorimetric Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or Calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells. For you question, to assess the growth at different time points e.g. 24,48,72 hours, and beyond, but without having to fix/freeze/kill the cells. It is advised to go ahead with Rapid Colorimetric Assay for cellular growth and survival. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. It can measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis. Ashish
I used CYQUANT (Molecular Probes) fluorescent based 96 well plate assay and aqueous one solution from Promega (optical density). Both worked well for me
I used CYQUANT (Molecular Probes) fluorescent based 96 well plate assay and aqueous one solution from Promega (optical density). Both worked well for me
More
VOTE
Cell titer blue or alamar blue can be used for this. It's a non-toxic dye and travels across the cell membrane so no fixing or lysing is necessary, but you have to be sure you thoroughly rinse afterwards (several media changes should be fine) and use the same protocol, gain, etc.
Cell titer blue or alamar blue can be used for this. It's a non-toxic dye and travels across the cell membrane so no fixing or lysing is necessary, but you have to be sure you thoroughly rinse afterwards (several media changes should be fine) and use the same protocol, gain, etc.
More
VOTE
For these type of experiments we usually use CellTiter Proliferation Assay. https://www.promega.de/products/cell-health-and-metabolism/cell-viability-assays/celltiter-96-aqueous-one-solution-cell-proliferation-assay-_mts_/?origUrl=http%3a%2f%2fwww.promega.com%3a81%2fproducts%2fcell-health-and-metabolism%2fcell-viability-assays%2fcelltiter-96-aqueous-one-solution-cell-proliferation-assay-_mts_%2f It is an easy to do colorimetric method. The Assays are performed by adding a small amount of the CellTiter directly to culture wells, incubating for 1–4 hours and then recording mitochondrial activity by absorbance at 490nm with a 96-well plate reader. The quantity of formazan product as measured by the amount of 490nm absorbance is directly proportional to the number of living cells in culture.
Another method, which we use in case the CellTiter Reagent is crossreacting with polymers or scaffolds is neutral red. https://www.researchgate.net/publication/5251539_Neutral_red_uptake_assay_for_the_estimation_of_cell_viabilitycytotoxicity
The neutral red uptake assay is based on the ability of viable cells to incorporate and bind neutral red in their lysosomes. It is also easy to do: Cells are incubated for 4 h with a medium containing 0.05% neutral red. The cells were subsequently washed with PBS. Afterwards, the cells are checked under the microscope and then the dye is extracted with (100 μl) desorb solution in each well and the absorbance was read using the 96 well-plate reader (Anthos, Microsystem, Germany) at 540 nm. Desorb solution: 50% Ethanol, 49% H2O, 1% Acid Acetic. As it is suggested by Repetto et al. 2008. I recommend to perform a standard curve for both assays.
Article Repetto G, del Peso A, Zurita JL.. Neutral red uptake assay ...
For these type of experiments we usually use CellTiter Proliferation Assay. https://www.promega.de/products/cell-health-and-metabolism/cell-viability-assays/celltiter-96-aqueous-one-solution-cell-proliferation-assay-_mts_/?origUrl=http%3a%2f%2fwww.promega.com%3a81%2fproducts%2fcell-health-and-metabolism%2fcell-viability-assays%2fcelltiter-96-aqueous-one-solution-cell-proliferation-assay-_mts_%2f It is an easy to do colorimetric method. The Assays are performed by adding a small amount of the CellTiter directly to culture wells, incubating for 1–4 hours and then recording mitochondrial activity by absorbance at 490nm with a 96-well plate reader. The quantity of formazan product as measured by the amount of 490nm absorbance is directly proportional to the number of living cells in culture.
Another method, which we use in case the CellTiter Reagent is crossreacting with polymers or scaffolds is neutral red. https://www.researchgate.net/publication/5251539_Neutral_red_uptake_assay_for_the_estimation_of_cell_viabilitycytotoxicity
The neutral red uptake assay is based on the ability of viable cells to incorporate and bind neutral red in their lysosomes. It is also easy to do: Cells are incubated for 4 h with a medium containing 0.05% neutral red. The cells were subsequently washed with PBS. Afterwards, the cells are checked under the microscope and then the dye is extracted with (100 μl) desorb solution in each well and the absorbance was read using the 96 well-plate reader (Anthos, Microsystem, Germany) at 540 nm. Desorb solution: 50% Ethanol, 49% H2O, 1% Acid Acetic. As it is suggested by Repetto et al. 2008. I recommend to perform a standard curve for both assays.
Article Repetto G, del Peso A, Zurita JL.. Neutral red uptake assay ...
More
VOTE
To work without dye or other chemical, we use the x-celligence system that report in real time the proliferation of adherent cells. It works with plates lined with an electrode measuring electrical impedance that estimates the surface of the wells occupied by cells.
http://www.aceabio.com/product.aspx
You can then have proliferation curves for multiple wells in real time without any chemicals.
Although you have to seed the cells directly on the plate (not on a coverslip), you might want to check if it is possible to place a matrix between the cells and the bottom of the well. Plates with "windows" are available: a part of the bottom's surface is devoid of electrode, this way you can monitor either cell morphology or staining of the cells (we do immunofluorescence).
To work without dye or other chemical, we use the x-celligence system that report in real time the proliferation of adherent cells. It works with plates lined with an electrode measuring electrical impedance that estimates the surface of the wells occupied by cells.
http://www.aceabio.com/product.aspx
You can then have proliferation curves for multiple wells in real time without any chemicals.
Although you have to seed the cells directly on the plate (not on a coverslip), you might want to check if it is possible to place a matrix between the cells and the bottom of the well. Plates with "windows" are available: a part of the bottom's surface is devoid of electrode, this way you can monitor either cell morphology or staining of the cells (we do immunofluorescence).
More
VOTE
Hi, Christopfer, if you have a good fluoresent microscope, you can try couple of fluoresent marker relate with cell proliferation. Fluoresent dyes, as Daniel mention, is also a good idea. But in some case dyes may have an effect on cell prolifaretion, if you incubate it for a long time.
In ideal case you can create a tarcking system whcih can take images automatically once per 20-30 minutes, for example. If you have histone:GFP marler. you can easy quantifies cell divsion ratio, as well as cell cycle duration etc.
But you need to have a precise automatical tracking system..
Good luck!
Hi, Christopfer, if you have a good fluoresent microscope, you can try couple of fluoresent marker relate with cell proliferation. Fluoresent dyes, as Daniel mention, is also a good idea. But in some case dyes may have an effect on cell prolifaretion, if you incubate it for a long time.
In ideal case you can create a tarcking system whcih can take images automatically once per 20-30 minutes, for example. If you have histone:GFP marler. you can easy quantifies cell divsion ratio, as well as cell cycle duration etc.
But you need to have a precise automatical tracking system..
Good luck!
More
VOTE
Most quantitative assays for cell proliferation are based on end-point measurements (EdU or BrdU incorporation, Ki67 staining, etc). However, several alternatives have been developed to measure cell proliferation using non-toxic fluorescent cell dyes (see this poster from Nikon, http://www.nikon.com/products/instruments/lineup/bioscience/integrated/ct/pdf/Estimation_of_Stem_Cell_Numbers.pdf), holographic imaging (http://www.phiab.se/applications/live-cell-imaging), or the FUCCI system (http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-cycle/live-cell-imaging-of-cell-cycle-and-division.html). All these methods require some level of specialized microscopy, so translucency and intrinsic fluorescent properties of your polymers could be limiting.
If the latter case proves true, then you could probably adopt a high-throughput assay by lysis of your cells at different times, and quantifying total DNA by Cyquant on a plate reader. Would revert to end-point assays, but there may be no better option if your system is not compatible with microscopy.
Most quantitative assays for cell proliferation are based on end-point measurements (EdU or BrdU incorporation, Ki67 staining, etc). However, several alternatives have been developed to measure cell proliferation using non-toxic fluorescent cell dyes (see this poster from Nikon, http://www.nikon.com/products/instruments/lineup/bioscience/integrated/ct/pdf/Estimation_of_Stem_Cell_Numbers.pdf), holographic imaging (http://www.phiab.se/applications/live-cell-imaging), or the FUCCI system (http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-cycle/live-cell-imaging-of-cell-cycle-and-division.html). All these methods require some level of specialized microscopy, so translucency and intrinsic fluorescent properties of your polymers could be limiting.
If the latter case proves true, then you could probably adopt a high-throughput assay by lysis of your cells at different times, and quantifying total DNA by Cyquant on a plate reader. Would revert to end-point assays, but there may be no better option if your system is not compatible with microscopy.
More
VOTE
Dear Christopher Charles West,
BrdU, EdU, MTT, XTT, WST, Fluorescent Dye Proliferation Assays, trypan blue or Calcein-AM are popular Cell Proliferation Assay techniques. In my opinion, Rapid Colorimetric Assay might be a quick and simple assays method that would allow you to do this assessment at multiple time points in the same well.
Cell Proliferation
Proliferating cells incorporate the radioactive label into their nascent DNA, which can be washed, adhered to filters and then measured using a scintillation counter. Assays to measure cellular proliferation, cell viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. MTT Calorimetric Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or Calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells.
For you question, to assess the growth at different time points e.g. 24,48,72 hours, and beyond, but without having to fix/freeze/kill the cells.
It is advised to go ahead with Rapid Colorimetric Assay for cellular growth and survival. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. It can measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
Ashish
Dear Christopher Charles West,
BrdU, EdU, MTT, XTT, WST, Fluorescent Dye Proliferation Assays, trypan blue or Calcein-AM are popular Cell Proliferation Assay techniques. In my opinion, Rapid Colorimetric Assay might be a quick and simple assays method that would allow you to do this assessment at multiple time points in the same well.
Cell Proliferation
Proliferating cells incorporate the radioactive label into their nascent DNA, which can be washed, adhered to filters and then measured using a scintillation counter. Assays to measure cellular proliferation, cell viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. MTT Calorimetric Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or Calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells.
For you question, to assess the growth at different time points e.g. 24,48,72 hours, and beyond, but without having to fix/freeze/kill the cells.
It is advised to go ahead with Rapid Colorimetric Assay for cellular growth and survival. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. It can measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
Ashish
More
VOTE
Dear Christopher Charles West,
BrdU, EdU, MTT, XTT, WST, Fluorescent Dye Proliferation Assays, trypan blue or Calcein-AM are popular Cell Proliferation Assay techniques. In my opinion, Rapid Colorimetric Assay might be a quick and simple assays method that would allow you to do this assessment at multiple time points in the same well.
Cell Proliferation
Proliferating cells incorporate the radioactive label into their nascent DNA, which can be washed, adhered to filters and then measured using a scintillation counter. Assays to measure cellular proliferation, cell viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. MTT Calorimetric Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or Calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells.
For you question, to assess the growth at different time points e.g. 24,48,72 hours, and beyond, but without having to fix/freeze/kill the cells.
It is advised to go ahead with Rapid Colorimetric Assay for cellular growth and survival. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. It can measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
Ashish
Dear Christopher Charles West,
BrdU, EdU, MTT, XTT, WST, Fluorescent Dye Proliferation Assays, trypan blue or Calcein-AM are popular Cell Proliferation Assay techniques. In my opinion, Rapid Colorimetric Assay might be a quick and simple assays method that would allow you to do this assessment at multiple time points in the same well.
Cell Proliferation
Proliferating cells incorporate the radioactive label into their nascent DNA, which can be washed, adhered to filters and then measured using a scintillation counter. Assays to measure cellular proliferation, cell viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. MTT Calorimetric Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or Calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells.
For you question, to assess the growth at different time points e.g. 24,48,72 hours, and beyond, but without having to fix/freeze/kill the cells.
It is advised to go ahead with Rapid Colorimetric Assay for cellular growth and survival. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. It can measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
Ashish
More
VOTE