Home >
Community >
How can I determine the NO production in THP-1 cells induced by LPS?
Upvote
22
Downvote
+ Thp-1
+ Cell culture
+ Lipopolysaccharides
+ Lps
+ Nitrous oxide
Posted by
David J. Hughes
How can I determine the NO production in THP-1 cells induced by LPS?
This is my protocol for cheking NO. I usi fresh Macrophage recruted from mice 4 days post IP injection with Thioglycollate 4%. I use 10 µg/ml LPS as activator. NO
Incubate cells for 24 hours at concentration of 〖1.5∙106〗cells/ml with desired reactors.
Centrifuge (spin-down) the cells at 1250rpm for 5 min.
Collect 50µl of medium for NO test into new wells.
Suspend medium 1:1 with Greiss Riagent (Sigma) for 15 minutes hidden from light.
This is my protocol for cheking NO. I usi fresh Macrophage recruted from mice 4 days post IP injection with Thioglycollate 4%. I use 10 µg/ml LPS as activator. NO
Incubate cells for 24 hours at concentration of 〖1.5∙106〗cells/ml with desired reactors.
Centrifuge (spin-down) the cells at 1250rpm for 5 min.
Collect 50µl of medium for NO test into new wells.
Suspend medium 1:1 with Greiss Riagent (Sigma) for 15 minutes hidden from light.
First, human macrophages are not robust NO producers in vitro (as compared to mouse cells for example), in fact the concept of NO production by human macrophages was quite controversial as recently as 10-12 years ago. Second, you'll need to provide a lot more information regarding the culture, experimental conditions, and assay conditions in order to obtain any help. Are you using differentiated cells? Say PMA stimulated? What passage? What concentration of LPS? What type? Have you tried a nitrite spike test into your cell culture media to see if you recover the correct amount of Nitrite and there is no interference? You can try co-stimulating with INF-gamma as well or supplement media with BH4.
First, human macrophages are not robust NO producers in vitro (as compared to mouse cells for example), in fact the concept of NO production by human macrophages was quite controversial as recently as 10-12 years ago. Second, you'll need to provide a lot more information regarding the culture, experimental conditions, and assay conditions in order to obtain any help. Are you using differentiated cells? Say PMA stimulated? What passage? What concentration of LPS? What type? Have you tried a nitrite spike test into your cell culture media to see if you recover the correct amount of Nitrite and there is no interference? You can try co-stimulating with INF-gamma as well or supplement media with BH4.
THP1 cells need to be differentiated to a mature phenotype for being able to detect NO. I recommend to use the murine RAW 264.7 macrophages instead and measuring NO secretion using Griess reagent.
THP1 cells need to be differentiated to a mature phenotype for being able to detect NO. I recommend to use the murine RAW 264.7 macrophages instead and measuring NO secretion using Griess reagent.
thanks all of you for your reply. some informartion of this experiment is that,THP-1 cells concentration is 0,875*106, After 24h incubated with 1ug/ml LPS,the plates were centrifuged (1200rmp,4°C,10mins),50ul cells culture supenatants in each well,then,determinate by Giess recation system.
thanks all of you for your reply. some informartion of this experiment is that,THP-1 cells concentration is 0,875*106, After 24h incubated with 1ug/ml LPS,the plates were centrifuged (1200rmp,4°C,10mins),50ul cells culture supenatants in each well,then,determinate by Giess recation system.
Sometimes, there is loss of a LBP (Lipase binding protein) or TLR4 (Toll like receptor 4) during the passaging, harvesting and /or subculturing which does not produce satisfactory results. Also, check the source of LPS (Sigma Aldrich is best). I agree with the aforesaid replies by others. RAW 264.7/J774A.1 cells are the suitable macrophages and co-administration of IFN-gamma/PMA +LPS also produce significant results.
Sometimes, there is loss of a LBP (Lipase binding protein) or TLR4 (Toll like receptor 4) during the passaging, harvesting and /or subculturing which does not produce satisfactory results. Also, check the source of LPS (Sigma Aldrich is best). I agree with the aforesaid replies by others. RAW 264.7/J774A.1 cells are the suitable macrophages and co-administration of IFN-gamma/PMA +LPS also produce significant results.
THP-1 cells do not produce any detectable nitric oxide in vitro even when differentiated into macrophages. The Griess reaction will not detect NO in THP-1 cells. However, you can implement EPR (electron paramagnetic resonance) where you can detect nitric oxide adducts using specific spin traps. This complex procedure detect nitric oxide release as a free radical unlike the Griess reaction that detect nitrite.
THP-1 cells do not produce any detectable nitric oxide in vitro even when differentiated into macrophages. The Griess reaction will not detect NO in THP-1 cells. However, you can implement EPR (electron paramagnetic resonance) where you can detect nitric oxide adducts using specific spin traps. This complex procedure detect nitric oxide release as a free radical unlike the Griess reaction that detect nitrite.
I also want to determine the NOx level in THP1 culture. we seeded 1x10^6 THP1 cells in 6-well plate and treated it with 100ng/mL PMA for 24h (M0) followed by 15ng/mL LPS and 50ng/mL IFN-g (M1) for macrophages differentiation. we harvested media at 0h, 24h, 48h, & 72h and measured NOx by Griess assay. I didn't find a difference in NOx level produced by M0 (PMA) and M1 (PMA followed by LPS and IFN-g). Even blank (RPMI media with 10% FBS have almost similar NOx concentration. How could I treat my cells (THP1) for biological NOx production? I will appreciate your suggestion. Faizan
I also want to determine the NOx level in THP1 culture. we seeded 1x10^6 THP1 cells in 6-well plate and treated it with 100ng/mL PMA for 24h (M0) followed by 15ng/mL LPS and 50ng/mL IFN-g (M1) for macrophages differentiation. we harvested media at 0h, 24h, 48h, & 72h and measured NOx by Griess assay. I didn't find a difference in NOx level produced by M0 (PMA) and M1 (PMA followed by LPS and IFN-g). Even blank (RPMI media with 10% FBS have almost similar NOx concentration. How could I treat my cells (THP1) for biological NOx production? I will appreciate your suggestion. Faizan
I am aware of the debate too and have decided to try to find ways to get human macrophages to produce NO. Currently, I cannot get 3day PMA-differentiated THP-1 cells to produce NO when using LPS (1ug/ml) and hIFNg (100U/ml), I have done measurements at 24, 48 and 72hr. DAFDA was used as a probe. I am currently considering trying other ways to stimulate NO production by adding TNFa.
I am aware of the debate too and have decided to try to find ways to get human macrophages to produce NO. Currently, I cannot get 3day PMA-differentiated THP-1 cells to produce NO when using LPS (1ug/ml) and hIFNg (100U/ml), I have done measurements at 24, 48 and 72hr. DAFDA was used as a probe. I am currently considering trying other ways to stimulate NO production by adding TNFa.
This is my protocol for cheking NO.
I usi fresh Macrophage recruted from mice 4 days post IP injection with Thioglycollate 4%.
I use 10 µg/ml LPS as activator.
NO
This is my protocol for cheking NO.
I usi fresh Macrophage recruted from mice 4 days post IP injection with Thioglycollate 4%.
I use 10 µg/ml LPS as activator.
NO
More
VOTE
First, human macrophages are not robust NO producers in vitro (as compared to mouse cells for example), in fact the concept of NO production by human macrophages was quite controversial as recently as 10-12 years ago.
Second, you'll need to provide a lot more information regarding the culture, experimental conditions, and assay conditions in order to obtain any help.
Are you using differentiated cells? Say PMA stimulated?
What passage?
What concentration of LPS? What type?
Have you tried a nitrite spike test into your cell culture media to see if you recover the correct amount of Nitrite and there is no interference?
You can try co-stimulating with INF-gamma as well or supplement media with BH4.
First, human macrophages are not robust NO producers in vitro (as compared to mouse cells for example), in fact the concept of NO production by human macrophages was quite controversial as recently as 10-12 years ago.
Second, you'll need to provide a lot more information regarding the culture, experimental conditions, and assay conditions in order to obtain any help.
Are you using differentiated cells? Say PMA stimulated?
What passage?
What concentration of LPS? What type?
Have you tried a nitrite spike test into your cell culture media to see if you recover the correct amount of Nitrite and there is no interference?
You can try co-stimulating with INF-gamma as well or supplement media with BH4.
More
VOTE
take a look to this work
http://www.sfrbm.org/pdf/Nitric-oxide_Grisham_FRBM2007.pdf
take a look to this work
http://www.sfrbm.org/pdf/Nitric-oxide_Grisham_FRBM2007.pdf
More
VOTE
THP1 cells need to be differentiated to a mature phenotype for being able to detect NO. I recommend to use the murine RAW 264.7 macrophages instead and measuring NO secretion using Griess reagent.
THP1 cells need to be differentiated to a mature phenotype for being able to detect NO. I recommend to use the murine RAW 264.7 macrophages instead and measuring NO secretion using Griess reagent.
More
VOTE
I also did not detected the NO production in LPS (1μg/ml, 12h)-induced THP-1 cell
I also did not detected the NO production in LPS (1μg/ml, 12h)-induced THP-1 cell
More
VOTE
thanks all of you for your reply.
some informartion of this experiment is that,THP-1 cells concentration is 0,875*106, After 24h incubated with 1ug/ml LPS,the plates were centrifuged (1200rmp,4°C,10mins),50ul cells culture supenatants in each well,then,determinate by Giess recation system.
thanks all of you for your reply.
some informartion of this experiment is that,THP-1 cells concentration is 0,875*106, After 24h incubated with 1ug/ml LPS,the plates were centrifuged (1200rmp,4°C,10mins),50ul cells culture supenatants in each well,then,determinate by Giess recation system.
More
VOTE
Sometimes, there is loss of a LBP (Lipase binding protein) or TLR4 (Toll like receptor 4) during the passaging, harvesting and /or subculturing which does not produce satisfactory results. Also, check the source of LPS (Sigma Aldrich is best). I agree with the aforesaid replies by others. RAW 264.7/J774A.1 cells are the suitable macrophages and co-administration of IFN-gamma/PMA +LPS also produce significant results.
Sometimes, there is loss of a LBP (Lipase binding protein) or TLR4 (Toll like receptor 4) during the passaging, harvesting and /or subculturing which does not produce satisfactory results. Also, check the source of LPS (Sigma Aldrich is best). I agree with the aforesaid replies by others. RAW 264.7/J774A.1 cells are the suitable macrophages and co-administration of IFN-gamma/PMA +LPS also produce significant results.
More
VOTE
THP-1 cells do not produce any detectable nitric oxide in vitro even when differentiated into macrophages. The Griess reaction will not detect NO in THP-1 cells. However, you can implement EPR (electron paramagnetic resonance) where you can detect nitric oxide adducts using specific spin traps. This complex procedure detect nitric oxide release as a free radical unlike the Griess reaction that detect nitrite.
THP-1 cells do not produce any detectable nitric oxide in vitro even when differentiated into macrophages. The Griess reaction will not detect NO in THP-1 cells. However, you can implement EPR (electron paramagnetic resonance) where you can detect nitric oxide adducts using specific spin traps. This complex procedure detect nitric oxide release as a free radical unlike the Griess reaction that detect nitrite.
More
VOTE
Who can tell me, at what concentration is the sigma greiss reagent prepared?
Who can tell me, at what concentration is the sigma greiss reagent prepared?
More
VOTE
I also want to determine the NOx level in THP1 culture. we seeded 1x10^6 THP1 cells in 6-well plate and treated it with 100ng/mL PMA for 24h (M0) followed by 15ng/mL LPS and 50ng/mL IFN-g (M1) for macrophages differentiation. we harvested media at 0h, 24h, 48h, & 72h and measured NOx by Griess assay. I didn't find a difference in NOx level produced by M0 (PMA) and M1 (PMA followed by LPS and IFN-g). Even blank (RPMI media with 10% FBS have almost similar NOx concentration.
How could I treat my cells (THP1) for biological NOx production?
I will appreciate your suggestion.
Faizan
I also want to determine the NOx level in THP1 culture. we seeded 1x10^6 THP1 cells in 6-well plate and treated it with 100ng/mL PMA for 24h (M0) followed by 15ng/mL LPS and 50ng/mL IFN-g (M1) for macrophages differentiation. we harvested media at 0h, 24h, 48h, & 72h and measured NOx by Griess assay. I didn't find a difference in NOx level produced by M0 (PMA) and M1 (PMA followed by LPS and IFN-g). Even blank (RPMI media with 10% FBS have almost similar NOx concentration.
How could I treat my cells (THP1) for biological NOx production?
I will appreciate your suggestion.
Faizan
More
VOTE
I am aware of the debate too and have decided to try to find ways to get human macrophages to produce NO. Currently, I cannot get 3day PMA-differentiated THP-1 cells to produce NO when using LPS (1ug/ml) and hIFNg (100U/ml), I have done measurements at 24, 48 and 72hr. DAFDA was used as a probe. I am currently considering trying other ways to stimulate NO production by adding TNFa.
I am aware of the debate too and have decided to try to find ways to get human macrophages to produce NO. Currently, I cannot get 3day PMA-differentiated THP-1 cells to produce NO when using LPS (1ug/ml) and hIFNg (100U/ml), I have done measurements at 24, 48 and 72hr. DAFDA was used as a probe. I am currently considering trying other ways to stimulate NO production by adding TNFa.
More
VOTE