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THP-1 to differentiated macrophages mostly dead after detachment...
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+ Thp-1
+ Macrophage
+ Trypsin
+ Thp-1 cells
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Michael Young
THP-1 to differentiated macrophages mostly dead after detachment...
It sounds to me that it's the LPS+INF treatment that's killing the cells so I'd suggest doing a concentration curve for both stimuli (first one, then the other) and check if you can get your differentiation markers right with a lower amount of either one.
The other suggestion would be indeed to try a different harvesting technique, you could for example try discarding all but a few ml of media, then firmly scrape once each area of the plate and leave the detached cells in the media (that works for me with MDMs) or pre-chill the plate on ice and then scrape.
It sounds to me that it's the LPS+INF treatment that's killing the cells so I'd suggest doing a concentration curve for both stimuli (first one, then the other) and check if you can get your differentiation markers right with a lower amount of either one.
The other suggestion would be indeed to try a different harvesting technique, you could for example try discarding all but a few ml of media, then firmly scrape once each area of the plate and leave the detached cells in the media (that works for me with MDMs) or pre-chill the plate on ice and then scrape.
Agree with Vinod Nadella's suggestions. You do not need to detach the PMA-treated THP-1 cells when you treat them with LPS and INF. I think this is the main problem.
Agree with Vinod Nadella's suggestions. You do not need to detach the PMA-treated THP-1 cells when you treat them with LPS and INF. I think this is the main problem.
Try incubating THP-1 cells with 150 nM PMA for 24 h in RPMI followed by 24 h incubation in RPMI medium alone. PMA at higher concentrations itself will differentiate the cells M1 and morphologically like M2. For polarizing to M1 phenotype incubation with 20 ng/ml of IFN-γ and 10 pg/ml of LPS. These concentrations and time frame is more than enough to kick them to your required phenotype. LPS is toxic and itself is good enough to kill the cells
I don't think there is a need to use Trypsin-EDTA and/or Accutase to detach them. Macrophages once differentiated are terminally differentiated and there won't be any change in their number as proliferations halts. Moreover,people loose the cells in culture especially in this process which depends from hand to hand.
The way the cells are handled matters as stress kills the cells. As these cells are pretty tricky to handle especially while trying to polarize to defined phenotype, gentle handing will help.
Try incubating THP-1 cells with 150 nM PMA for 24 h in RPMI followed by 24 h incubation in RPMI medium alone. PMA at higher concentrations itself will differentiate the cells M1 and morphologically like M2. For polarizing to M1 phenotype incubation with 20 ng/ml of IFN-γ and 10 pg/ml of LPS. These concentrations and time frame is more than enough to kick them to your required phenotype. LPS is toxic and itself is good enough to kill the cells
I don't think there is a need to use Trypsin-EDTA and/or Accutase to detach them. Macrophages once differentiated are terminally differentiated and there won't be any change in their number as proliferations halts. Moreover,people loose the cells in culture especially in this process which depends from hand to hand.
The way the cells are handled matters as stress kills the cells. As these cells are pretty tricky to handle especially while trying to polarize to defined phenotype, gentle handing will help.
From my experience, once THP-1 monocytes cells are differentiated using PMA to M0 macrophage cells they do not further multiply. Therefore, you do not need to detach the differentiated macrophage and re-seed the amount you want for polarizing to M1. Just straight away differentiated the number of cells you needed from monocyte stage to M0 macrophage state and polarize them to M1 state.
From my experience, once THP-1 monocytes cells are differentiated using PMA to M0 macrophage cells they do not further multiply. Therefore, you do not need to detach the differentiated macrophage and re-seed the amount you want for polarizing to M1. Just straight away differentiated the number of cells you needed from monocyte stage to M0 macrophage state and polarize them to M1 state.
Agree with Laura Díaz-Alvarez's suggestions to 1) Titre LPS+INF concentrations. Ideally even with 10 % V/V serum, 10 ng/ul is LPS is enough for me. 2) Modify or change your harvesting procedure by using different cell detachment methods like i) Leave 1-2 ml medium (or PBS) in the plate and scrape off cells gently and slowly with rubber policeman blade ( cell scraper) at ~45 degree angel. ii) Alternative method is to mature THP to macrophages in Teflon containers. Cells do have typical macrophage phenotype and don't adhere to Teflon. iii) Incubation with non enzymatic cell dissociation solution ( SIGMA-based on a PBS EDTA solution) then very gently scraping the cells using a cell scraper. I dea here is to get cells out using different techniques and asses viability downstream post treatment with LPS+IFN.
Agree with Laura Díaz-Alvarez's suggestions to 1) Titre LPS+INF concentrations. Ideally even with 10 % V/V serum, 10 ng/ul is LPS is enough for me. 2) Modify or change your harvesting procedure by using different cell detachment methods like i) Leave 1-2 ml medium (or PBS) in the plate and scrape off cells gently and slowly with rubber policeman blade ( cell scraper) at ~45 degree angel. ii) Alternative method is to mature THP to macrophages in Teflon containers. Cells do have typical macrophage phenotype and don't adhere to Teflon. iii) Incubation with non enzymatic cell dissociation solution ( SIGMA-based on a PBS EDTA solution) then very gently scraping the cells using a cell scraper. I dea here is to get cells out using different techniques and asses viability downstream post treatment with LPS+IFN.
It sounds to me that it's the LPS+INF treatment that's killing the cells so I'd suggest doing a concentration curve for both stimuli (first one, then the other) and check if you can get your differentiation markers right with a lower amount of either one.
The other suggestion would be indeed to try a different harvesting technique, you could for example try discarding all but a few ml of media, then firmly scrape once each area of the plate and leave the detached cells in the media (that works for me with MDMs) or pre-chill the plate on ice and then scrape.
Here's a video that might help
https://youtu.be/5_LK5XCRTMU
It sounds to me that it's the LPS+INF treatment that's killing the cells so I'd suggest doing a concentration curve for both stimuli (first one, then the other) and check if you can get your differentiation markers right with a lower amount of either one.
The other suggestion would be indeed to try a different harvesting technique, you could for example try discarding all but a few ml of media, then firmly scrape once each area of the plate and leave the detached cells in the media (that works for me with MDMs) or pre-chill the plate on ice and then scrape.
Here's a video that might help
https://youtu.be/5_LK5XCRTMU
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Agree with Vinod Nadella's suggestions. You do not need to detach the PMA-treated THP-1 cells when you treat them with LPS and INF. I think this is the main problem.
Agree with Vinod Nadella's suggestions. You do not need to detach the PMA-treated THP-1 cells when you treat them with LPS and INF. I think this is the main problem.
More
VOTE
Try incubating THP-1 cells with 150 nM PMA for 24 h in RPMI followed by 24 h incubation in RPMI medium alone. PMA at higher concentrations itself will differentiate the cells M1 and morphologically like M2. For polarizing to M1 phenotype incubation with 20 ng/ml of IFN-γ and 10 pg/ml of LPS. These concentrations and time frame is more than enough to kick them to your required phenotype. LPS is toxic and itself is good enough to kill the cells
I don't think there is a need to use Trypsin-EDTA and/or Accutase to detach them. Macrophages once differentiated are terminally differentiated and there won't be any change in their number as proliferations halts. Moreover,people loose the cells in culture especially in this process which depends from hand to hand.
The way the cells are handled matters as stress kills the cells. As these cells are pretty tricky to handle especially while trying to polarize to defined phenotype, gentle handing will help.
BW
Try incubating THP-1 cells with 150 nM PMA for 24 h in RPMI followed by 24 h incubation in RPMI medium alone. PMA at higher concentrations itself will differentiate the cells M1 and morphologically like M2. For polarizing to M1 phenotype incubation with 20 ng/ml of IFN-γ and 10 pg/ml of LPS. These concentrations and time frame is more than enough to kick them to your required phenotype. LPS is toxic and itself is good enough to kill the cells
I don't think there is a need to use Trypsin-EDTA and/or Accutase to detach them. Macrophages once differentiated are terminally differentiated and there won't be any change in their number as proliferations halts. Moreover,people loose the cells in culture especially in this process which depends from hand to hand.
The way the cells are handled matters as stress kills the cells. As these cells are pretty tricky to handle especially while trying to polarize to defined phenotype, gentle handing will help.
BW
More
VOTE
From my experience, once THP-1 monocytes cells are differentiated using PMA to M0 macrophage cells they do not further multiply. Therefore, you do not need to detach the differentiated macrophage and re-seed the amount you want for polarizing to M1. Just straight away differentiated the number of cells you needed from monocyte stage to M0 macrophage state and polarize them to M1 state.
From my experience, once THP-1 monocytes cells are differentiated using PMA to M0 macrophage cells they do not further multiply. Therefore, you do not need to detach the differentiated macrophage and re-seed the amount you want for polarizing to M1. Just straight away differentiated the number of cells you needed from monocyte stage to M0 macrophage state and polarize them to M1 state.
More
VOTE
Agree with Laura Díaz-Alvarez's suggestions to 1) Titre LPS+INF concentrations. Ideally even with 10 % V/V serum, 10 ng/ul is LPS is enough for me. 2) Modify or change your harvesting procedure by using different cell detachment methods like i) Leave 1-2 ml medium (or PBS) in the plate and scrape off cells gently and slowly with rubber policeman blade ( cell scraper) at ~45 degree angel. ii) Alternative method is to mature THP to macrophages in Teflon containers. Cells do have typical macrophage phenotype and don't adhere to Teflon. iii) Incubation with non enzymatic cell dissociation solution ( SIGMA-based on a PBS EDTA solution) then very gently scraping the cells using a cell scraper.
I dea here is to get cells out using different techniques and asses viability downstream post treatment with LPS+IFN.
Agree with Laura Díaz-Alvarez's suggestions to 1) Titre LPS+INF concentrations. Ideally even with 10 % V/V serum, 10 ng/ul is LPS is enough for me. 2) Modify or change your harvesting procedure by using different cell detachment methods like i) Leave 1-2 ml medium (or PBS) in the plate and scrape off cells gently and slowly with rubber policeman blade ( cell scraper) at ~45 degree angel. ii) Alternative method is to mature THP to macrophages in Teflon containers. Cells do have typical macrophage phenotype and don't adhere to Teflon. iii) Incubation with non enzymatic cell dissociation solution ( SIGMA-based on a PBS EDTA solution) then very gently scraping the cells using a cell scraper.
I dea here is to get cells out using different techniques and asses viability downstream post treatment with LPS+IFN.
More
VOTE