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What is the best way to measure ROS in macrophages?
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+ Flow cytometry
+ Dcfh-da assay
+ Reactive oxygen species
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Kevin Cottrell
What is the best way to measure ROS in macrophages?
Hello Nilanka! I´m using CellROX® Deep Red Reagent for oxidative stress detection in FC. I didn´t try it on plate reader. Cell fixation is not a prerequisite for that reagent. You should definitely be aware that the conversion by PBMC-derived Mphs is much more faster than cell lines. The kinetic measurement on FC gave first sign of substrate conversion in just 30 min. I used 30,000 cells and 200nM of the reagent. Make sure your cells are rinsed well before adding the substrate, because ROS is rapidly released in cell media. Good luck with this assay :)
Hello Nilanka! I´m using CellROX® Deep Red Reagent for oxidative stress detection in FC. I didn´t try it on plate reader. Cell fixation is not a prerequisite for that reagent. You should definitely be aware that the conversion by PBMC-derived Mphs is much more faster than cell lines. The kinetic measurement on FC gave first sign of substrate conversion in just 30 min. I used 30,000 cells and 200nM of the reagent. Make sure your cells are rinsed well before adding the substrate, because ROS is rapidly released in cell media. Good luck with this assay :)
In addition, I want to point to our recent paper, in which we investigated a new variant of non-canonical autophagy in macrophages infected with Listeria monocytogenes. Importantly, this paper also shows that not every macrophage type is suitable for every study and experimental analysis. We show that BMDM have much less of the ROS-generating enzyme Nox2 and therefore produce neearly no extracellular/phagosomal ROS. Only after pro-inflammatory priming, Nox2 protein levels and ROS production nearly reach levels of ex vivo peritoneal macrophages. If you like, please have a look at:
In addition, I want to point to our recent paper, in which we investigated a new variant of non-canonical autophagy in macrophages infected with Listeria monocytogenes. Importantly, this paper also shows that not every macrophage type is suitable for every study and experimental analysis. We show that BMDM have much less of the ROS-generating enzyme Nox2 and therefore produce neearly no extracellular/phagosomal ROS. Only after pro-inflammatory priming, Nox2 protein levels and ROS production nearly reach levels of ex vivo peritoneal macrophages. If you like, please have a look at:
Try to use DHE (dihydroethidium), I was using it for confocal microscopy, but it can also be used with flow cytometry. This protocol was very useful for me: http://www.nature.com/protocolexchange/protocols/414#/equipment Authors claim that: 'DHE is perhaps the most specific and least problematic dye; as it detects essentially superoxide radicals, is retained well by cells, and may even tolerate mild fixation.' Hope this helps
Try to use DHE (dihydroethidium), I was using it for confocal microscopy, but it can also be used with flow cytometry. This protocol was very useful for me: http://www.nature.com/protocolexchange/protocols/414#/equipment Authors claim that: 'DHE is perhaps the most specific and least problematic dye; as it detects essentially superoxide radicals, is retained well by cells, and may even tolerate mild fixation.' Hope this helps
In addition, I want to point to our recent paper, in which we investigated a new variant of non-canonical autophagy in macrophages infected with Listeria monocytogenes. Importantly, this paper also shows that not every macrophage type is suitable for every study and experimental analysis. We show that BMDM have much less of the ROS-generating enzyme Nox2 and therefore produce neearly no extracellular/phagosomal ROS. Only after pro-inflammatory priming, Nox2 protein levels and ROS production nearly reach levels of ex vivo peritoneal macrophages. If you like, please have a look at: ArticleMacrophages target Listeria monocytogenes by two discrete no...
In addition, I want to point to our recent paper, in which we investigated a new variant of non-canonical autophagy in macrophages infected with Listeria monocytogenes. Importantly, this paper also shows that not every macrophage type is suitable for every study and experimental analysis. We show that BMDM have much less of the ROS-generating enzyme Nox2 and therefore produce neearly no extracellular/phagosomal ROS. Only after pro-inflammatory priming, Nox2 protein levels and ROS production nearly reach levels of ex vivo peritoneal macrophages. If you like, please have a look at: ArticleMacrophages target Listeria monocytogenes by two discrete no...
Hi, U can do with flow cytometer as well with DCFH-DA dye. Remember ROS should be measured within 15-30 min after adding dye. No need to fix after using dye. just wash it then measure it.
Hi, U can do with flow cytometer as well with DCFH-DA dye. Remember ROS should be measured within 15-30 min after adding dye. No need to fix after using dye. just wash it then measure it.
In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".
In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).
If you like, please have a look at: Functions of ROS in Macrophages and Antimicrobial Immunity February 2021, Antioxidants 10(2):313 DOI: https://doi.org/10.3390/antiox10020313
In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".
In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).
If you like, please have a look at: Functions of ROS in Macrophages and Antimicrobial Immunity February 2021, Antioxidants 10(2):313 DOI: https://doi.org/10.3390/antiox10020313
ROS measurements are done routinely in our lab mainly with different ex vivo cells from the innate immune system, but it took a long time to set it right.
One of my concerns about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics instead of making "ROS-Snapshots". Moreover you do not encounter the problem of fixating the ROS probes.
There are a lot of fancy new and very specific ROS probes (ex and in vivo) on the market and the development still goes on. However, for simple and easy-to-perfom ROS measuremtents, we established some protocols that work with 50.000-100.000 cells per well in a 96-well plate.
We sucessfully measured ROS with our commonly used ROS probes (Isoluminol for extracellular ROS, 6,5-Dicarboxy-DCF for cystosolicROS and MitoSOX for MitoMatrix ROS) with bacterial infection and this worked quite well. As chemical positive controls for Isoluminol and DCF measurements we use PMA (activates a lot in cells, so if your cells do not prodcue ROS after PMA, it is very likely they will also not produce ROS with your physiological stimulus) and for MitoSOX we use rotenone (blocks electron flow from complex I to complex III of the electron transport chain, electrons flow back into the matrix and ROS are formed).
There are ROS probes, wich I would not recommend. The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA and for CellROX, which a lot of people use. This also freely diffuses over membranes, so with these probes you only detect "overall ROS" in the cell.
For ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF). The protocol for Isoluminol also works fine with Amplex Red (measures specifically extracellular H2O2)
For any questions, informations or technical details for ROS measurements and how these measurements look in a plate reader (in macrophages with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 70 % of it is more or less about ROS measurements in a plate reader.
The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
ROS measurements are done routinely in our lab mainly with different ex vivo cells from the innate immune system, but it took a long time to set it right.
One of my concerns about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics instead of making "ROS-Snapshots". Moreover you do not encounter the problem of fixating the ROS probes.
There are a lot of fancy new and very specific ROS probes (ex and in vivo) on the market and the development still goes on. However, for simple and easy-to-perfom ROS measuremtents, we established some protocols that work with 50.000-100.000 cells per well in a 96-well plate.
We sucessfully measured ROS with our commonly used ROS probes (Isoluminol for extracellular ROS, 6,5-Dicarboxy-DCF for cystosolicROS and MitoSOX for MitoMatrix ROS) with bacterial infection and this worked quite well. As chemical positive controls for Isoluminol and DCF measurements we use PMA (activates a lot in cells, so if your cells do not prodcue ROS after PMA, it is very likely they will also not produce ROS with your physiological stimulus) and for MitoSOX we use rotenone (blocks electron flow from complex I to complex III of the electron transport chain, electrons flow back into the matrix and ROS are formed).
There are ROS probes, wich I would not recommend. The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA and for CellROX, which a lot of people use. This also freely diffuses over membranes, so with these probes you only detect "overall ROS" in the cell.
For ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF). The protocol for Isoluminol also works fine with Amplex Red (measures specifically extracellular H2O2)
For any questions, informations or technical details for ROS measurements and how these measurements look in a plate reader (in macrophages with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 70 % of it is more or less about ROS measurements in a plate reader.
The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
ROS measurements are done routinely in our lab mainly with different ex vivo cells from the innate immune system, but it took a long time to set it right.
One of my concerns about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics instead of making "ROS-Snapshots". Moreover you do not encounter the problem of fixating the ROS probes.
There are a lot of fancy new and very specific ROS probes (ex and in vivo) on the market and the development still goes on. However, for simple and easy-to-perfom ROS measuremtents, we established some protocols that work with 50.000-100.000 cells per well in a 96-well plate.
We sucessfully measured ROS with our commonly used ROS probes (Isoluminol for extracellular ROS, 6,5-Dicarboxy-DCF for cystosolicROS and MitoSOX for MitoMatrix ROS) with bacterial infection and this worked quite well. As chemical positive controls for Isoluminol and DCF measurements we use PMA (activates a lot in cells, so if your cells do not prodcue ROS after PMA, it is very likely they will also not produce ROS with your physiological stimulus) and for MitoSOX we use rotenone (blocks electron flow from complex I to complex III of the electron transport chain, electrons flow back into the matrix and ROS are formed).
There are ROS probes, wich I would not recommend. The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA and for CellROX, which a lot of people use. This also freely diffuses over membranes, so with these probes you only detect "overall ROS" in the cell.
For ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF). The protocol for Isoluminol also works fine with Amplex Red (measures specifically extracellular H2O2)
For any questions, informations or technical details for ROS measurements and how these measurements look in a plate reader (in macrophages with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 70 % of it is more or less about ROS measurements in a plate reader.
The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
ROS measurements are done routinely in our lab mainly with different ex vivo cells from the innate immune system, but it took a long time to set it right.
One of my concerns about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics instead of making "ROS-Snapshots". Moreover you do not encounter the problem of fixating the ROS probes.
There are a lot of fancy new and very specific ROS probes (ex and in vivo) on the market and the development still goes on. However, for simple and easy-to-perfom ROS measuremtents, we established some protocols that work with 50.000-100.000 cells per well in a 96-well plate.
We sucessfully measured ROS with our commonly used ROS probes (Isoluminol for extracellular ROS, 6,5-Dicarboxy-DCF for cystosolicROS and MitoSOX for MitoMatrix ROS) with bacterial infection and this worked quite well. As chemical positive controls for Isoluminol and DCF measurements we use PMA (activates a lot in cells, so if your cells do not prodcue ROS after PMA, it is very likely they will also not produce ROS with your physiological stimulus) and for MitoSOX we use rotenone (blocks electron flow from complex I to complex III of the electron transport chain, electrons flow back into the matrix and ROS are formed).
There are ROS probes, wich I would not recommend. The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA and for CellROX, which a lot of people use. This also freely diffuses over membranes, so with these probes you only detect "overall ROS" in the cell.
For ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF). The protocol for Isoluminol also works fine with Amplex Red (measures specifically extracellular H2O2)
For any questions, informations or technical details for ROS measurements and how these measurements look in a plate reader (in macrophages with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 70 % of it is more or less about ROS measurements in a plate reader.
The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
Thank you everyone. Very helpful. And thank you again for the papers shared. I need to fix my cells as DENV is CAT3 and our Flow cytometer is in CAT2. I will let you know what happens!
Thank you everyone. Very helpful. And thank you again for the papers shared. I need to fix my cells as DENV is CAT3 and our Flow cytometer is in CAT2. I will let you know what happens!
Hello Nilanka! I´m using CellROX® Deep Red Reagent for oxidative stress detection in FC. I didn´t try it on plate reader. Cell fixation is not a prerequisite for that reagent. You should definitely be aware that the conversion by PBMC-derived Mphs is much more faster than cell lines. The kinetic measurement on FC gave first sign of substrate conversion in just 30 min. I used 30,000 cells and 200nM of the reagent. Make sure your cells are rinsed well before adding the substrate, because ROS is rapidly released in cell media. Good luck with this assay :)
Hello Nilanka! I´m using CellROX® Deep Red Reagent for oxidative stress detection in FC. I didn´t try it on plate reader. Cell fixation is not a prerequisite for that reagent. You should definitely be aware that the conversion by PBMC-derived Mphs is much more faster than cell lines. The kinetic measurement on FC gave first sign of substrate conversion in just 30 min. I used 30,000 cells and 200nM of the reagent. Make sure your cells are rinsed well before adding the substrate, because ROS is rapidly released in cell media. Good luck with this assay :)
More
VOTE
In addition, I want to point to our recent paper, in which we investigated a new variant of non-canonical autophagy in macrophages infected with Listeria monocytogenes. Importantly, this paper also shows that not every macrophage type is suitable for every study and experimental analysis. We show that BMDM have much less of the ROS-generating enzyme Nox2 and therefore produce neearly no extracellular/phagosomal ROS. Only after pro-inflammatory priming, Nox2 protein levels and ROS production nearly reach levels of ex vivo peritoneal macrophages.
If you like, please have a look at:
Article Macrophages target Listeria monocytogenes by two discrete no...
All the best and stay healthy,
Marc
In addition, I want to point to our recent paper, in which we investigated a new variant of non-canonical autophagy in macrophages infected with Listeria monocytogenes. Importantly, this paper also shows that not every macrophage type is suitable for every study and experimental analysis. We show that BMDM have much less of the ROS-generating enzyme Nox2 and therefore produce neearly no extracellular/phagosomal ROS. Only after pro-inflammatory priming, Nox2 protein levels and ROS production nearly reach levels of ex vivo peritoneal macrophages.
If you like, please have a look at:
Article Macrophages target Listeria monocytogenes by two discrete no...
All the best and stay healthy,
Marc
More
VOTE
Try to use DHE (dihydroethidium), I was using it for confocal microscopy, but it can also be used with flow cytometry. This protocol was very useful for me:
http://www.nature.com/protocolexchange/protocols/414#/equipment
Authors claim that: 'DHE is perhaps the most specific and least problematic dye; as it detects essentially superoxide radicals, is retained well by cells, and may even tolerate mild fixation.'
Hope this helps
Try to use DHE (dihydroethidium), I was using it for confocal microscopy, but it can also be used with flow cytometry. This protocol was very useful for me:
http://www.nature.com/protocolexchange/protocols/414#/equipment
Authors claim that: 'DHE is perhaps the most specific and least problematic dye; as it detects essentially superoxide radicals, is retained well by cells, and may even tolerate mild fixation.'
Hope this helps
More
VOTE
In addition, I want to point to our recent paper, in which we investigated a new variant of non-canonical autophagy in macrophages infected with Listeria monocytogenes. Importantly, this paper also shows that not every macrophage type is suitable for every study and experimental analysis. We show that BMDM have much less of the ROS-generating enzyme Nox2 and therefore produce neearly no extracellular/phagosomal ROS. Only after pro-inflammatory priming, Nox2 protein levels and ROS production nearly reach levels of ex vivo peritoneal macrophages.
If you like, please have a look at:
Article Macrophages target Listeria monocytogenes by two discrete no...
All the best and stay healthy,
Marc
In addition, I want to point to our recent paper, in which we investigated a new variant of non-canonical autophagy in macrophages infected with Listeria monocytogenes. Importantly, this paper also shows that not every macrophage type is suitable for every study and experimental analysis. We show that BMDM have much less of the ROS-generating enzyme Nox2 and therefore produce neearly no extracellular/phagosomal ROS. Only after pro-inflammatory priming, Nox2 protein levels and ROS production nearly reach levels of ex vivo peritoneal macrophages.
If you like, please have a look at:
Article Macrophages target Listeria monocytogenes by two discrete no...
All the best and stay healthy,
Marc
More
VOTE
Hi Nilanka,
I'm not sure what the active agent is of the ROS-ID™ Total ROS/Superoxide detection kit (Enzo) that we used when we studied dengue induced production of ROS (http://www.nature.com/cddis/journal/v7/n3/full/cddis2015409a.html OR https://www.researchgate.net/publication/296698867_Dengue-induced_autophagy_virus_replication_and_protection_from_cell_death_require_ER_stress_PERK_pathway_activation).
But it gave us very good signal especially with the positive control pyocyanin. I think it's a bit cheaper too. Hope this helps!
-ED
Article Dengue-induced autophagy, virus replication and protection f...
Hi Nilanka,
I'm not sure what the active agent is of the ROS-ID™ Total ROS/Superoxide detection kit (Enzo) that we used when we studied dengue induced production of ROS (http://www.nature.com/cddis/journal/v7/n3/full/cddis2015409a.html OR https://www.researchgate.net/publication/296698867_Dengue-induced_autophagy_virus_replication_and_protection_from_cell_death_require_ER_stress_PERK_pathway_activation).
But it gave us very good signal especially with the positive control pyocyanin. I think it's a bit cheaper too. Hope this helps!
-ED
Article Dengue-induced autophagy, virus replication and protection f...
More
VOTE
Please also have look at our Perspective article, if you like:
Article Reactive Oxygen Species: Not Omnipresent but Important in Ma...
Please also have look at our Perspective article, if you like:
Article Reactive Oxygen Species: Not Omnipresent but Important in Ma...
More
VOTE
Hi,
U can do with flow cytometer as well with DCFH-DA dye. Remember ROS should be measured within 15-30 min after adding dye. No need to fix after using dye. just wash it then measure it.
good luck
Hi,
U can do with flow cytometer as well with DCFH-DA dye. Remember ROS should be measured within 15-30 min after adding dye. No need to fix after using dye. just wash it then measure it.
good luck
More
VOTE
In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".
In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).
If you like, please have a look at:
Functions of ROS in Macrophages and Antimicrobial Immunity
February 2021, Antioxidants 10(2):313
DOI: https://doi.org/10.3390/antiox10020313
In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".
In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).
If you like, please have a look at:
Functions of ROS in Macrophages and Antimicrobial Immunity
February 2021, Antioxidants 10(2):313
DOI: https://doi.org/10.3390/antiox10020313
More
VOTE
Hey Nilanka,
ROS measurements are done routinely in our lab mainly with different ex vivo cells from the innate immune system, but it took a long time to set it right.
One of my concerns about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics instead of making "ROS-Snapshots". Moreover you do not encounter the problem of fixating the ROS probes.
There are a lot of fancy new and very specific ROS probes (ex and in vivo) on the market and the development still goes on. However, for simple and easy-to-perfom ROS measuremtents, we established some protocols that work with 50.000-100.000 cells per well in a 96-well plate.
We sucessfully measured ROS with our commonly used ROS probes (Isoluminol for extracellular ROS, 6,5-Dicarboxy-DCF for cystosolic ROS and MitoSOX for MitoMatrix ROS) with bacterial infection and this worked quite well. As chemical positive controls for Isoluminol and DCF measurements we use PMA (activates a lot in cells, so if your cells do not prodcue ROS after PMA, it is very likely they will also not produce ROS with your physiological stimulus) and for MitoSOX we use rotenone (blocks electron flow from complex I to complex III of the electron transport chain, electrons flow back into the matrix and ROS are formed).
There are ROS probes, wich I would not recommend.
The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA and for CellROX, which a lot of people use. This also freely diffuses over membranes, so with these probes you only detect "overall ROS" in the cell.
For ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF). The protocol for Isoluminol also works fine with Amplex Red (measures specifically extracellular H2O2)
For any questions, informations or technical details for ROS measurements and how these measurements look in a plate reader (in macrophages with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 70 % of it is more or less about ROS measurements in a plate reader.
The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
https://stke.sciencemag.org/content/12/568/eaar5926
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO. Science Signaling (2019)
Please feel free to ask any further questions or add your experiences.
All the best,
Marc
Hey Nilanka,
ROS measurements are done routinely in our lab mainly with different ex vivo cells from the innate immune system, but it took a long time to set it right.
One of my concerns about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics instead of making "ROS-Snapshots". Moreover you do not encounter the problem of fixating the ROS probes.
There are a lot of fancy new and very specific ROS probes (ex and in vivo) on the market and the development still goes on. However, for simple and easy-to-perfom ROS measuremtents, we established some protocols that work with 50.000-100.000 cells per well in a 96-well plate.
We sucessfully measured ROS with our commonly used ROS probes (Isoluminol for extracellular ROS, 6,5-Dicarboxy-DCF for cystosolic ROS and MitoSOX for MitoMatrix ROS) with bacterial infection and this worked quite well. As chemical positive controls for Isoluminol and DCF measurements we use PMA (activates a lot in cells, so if your cells do not prodcue ROS after PMA, it is very likely they will also not produce ROS with your physiological stimulus) and for MitoSOX we use rotenone (blocks electron flow from complex I to complex III of the electron transport chain, electrons flow back into the matrix and ROS are formed).
There are ROS probes, wich I would not recommend.
The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA and for CellROX, which a lot of people use. This also freely diffuses over membranes, so with these probes you only detect "overall ROS" in the cell.
For ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF). The protocol for Isoluminol also works fine with Amplex Red (measures specifically extracellular H2O2)
For any questions, informations or technical details for ROS measurements and how these measurements look in a plate reader (in macrophages with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 70 % of it is more or less about ROS measurements in a plate reader.
The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
https://stke.sciencemag.org/content/12/568/eaar5926
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO. Science Signaling (2019)
Please feel free to ask any further questions or add your experiences.
All the best,
Marc
More
VOTE
Hey Nilanka,
ROS measurements are done routinely in our lab mainly with different ex vivo cells from the innate immune system, but it took a long time to set it right.
One of my concerns about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics instead of making "ROS-Snapshots". Moreover you do not encounter the problem of fixating the ROS probes.
There are a lot of fancy new and very specific ROS probes (ex and in vivo) on the market and the development still goes on. However, for simple and easy-to-perfom ROS measuremtents, we established some protocols that work with 50.000-100.000 cells per well in a 96-well plate.
We sucessfully measured ROS with our commonly used ROS probes (Isoluminol for extracellular ROS, 6,5-Dicarboxy-DCF for cystosolic ROS and MitoSOX for MitoMatrix ROS) with bacterial infection and this worked quite well. As chemical positive controls for Isoluminol and DCF measurements we use PMA (activates a lot in cells, so if your cells do not prodcue ROS after PMA, it is very likely they will also not produce ROS with your physiological stimulus) and for MitoSOX we use rotenone (blocks electron flow from complex I to complex III of the electron transport chain, electrons flow back into the matrix and ROS are formed).
There are ROS probes, wich I would not recommend.
The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA and for CellROX, which a lot of people use. This also freely diffuses over membranes, so with these probes you only detect "overall ROS" in the cell.
For ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF). The protocol for Isoluminol also works fine with Amplex Red (measures specifically extracellular H2O2)
For any questions, informations or technical details for ROS measurements and how these measurements look in a plate reader (in macrophages with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 70 % of it is more or less about ROS measurements in a plate reader.
The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
https://stke.sciencemag.org/content/12/568/eaar5926
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO. Science Signaling (2019)
Please feel free to ask any further questions or add your experiences.
All the best,
Marc
Hey Nilanka,
ROS measurements are done routinely in our lab mainly with different ex vivo cells from the innate immune system, but it took a long time to set it right.
One of my concerns about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics instead of making "ROS-Snapshots". Moreover you do not encounter the problem of fixating the ROS probes.
There are a lot of fancy new and very specific ROS probes (ex and in vivo) on the market and the development still goes on. However, for simple and easy-to-perfom ROS measuremtents, we established some protocols that work with 50.000-100.000 cells per well in a 96-well plate.
We sucessfully measured ROS with our commonly used ROS probes (Isoluminol for extracellular ROS, 6,5-Dicarboxy-DCF for cystosolic ROS and MitoSOX for MitoMatrix ROS) with bacterial infection and this worked quite well. As chemical positive controls for Isoluminol and DCF measurements we use PMA (activates a lot in cells, so if your cells do not prodcue ROS after PMA, it is very likely they will also not produce ROS with your physiological stimulus) and for MitoSOX we use rotenone (blocks electron flow from complex I to complex III of the electron transport chain, electrons flow back into the matrix and ROS are formed).
There are ROS probes, wich I would not recommend.
The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA and for CellROX, which a lot of people use. This also freely diffuses over membranes, so with these probes you only detect "overall ROS" in the cell.
For ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF). The protocol for Isoluminol also works fine with Amplex Red (measures specifically extracellular H2O2)
For any questions, informations or technical details for ROS measurements and how these measurements look in a plate reader (in macrophages with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 70 % of it is more or less about ROS measurements in a plate reader.
The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
https://stke.sciencemag.org/content/12/568/eaar5926
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO. Science Signaling (2019)
Please feel free to ask any further questions or add your experiences.
All the best,
Marc
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Thank you everyone. Very helpful. And thank you again for the papers shared. I need to fix my cells as DENV is CAT3 and our Flow cytometer is in CAT2. I will let you know what happens!
Thank you everyone. Very helpful. And thank you again for the papers shared. I need to fix my cells as DENV is CAT3 and our Flow cytometer is in CAT2. I will let you know what happens!
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