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Problems with THP-1 cells. How to culture them?
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Marty Bannister
Problems with THP-1 cells. How to culture them?
Dear Edwardo, THP-1 has a doubling time of less than 20 hours. The problem lies in the seeding area of cells. THP-1 cells should be grown at a density of 1 million to 2 million/mL .In a 24 well 1 million cells could be easily seeded and grown( no differentiation). In a 250ml culture flask the maximum media should be 50mL and between 20-30 million for seeding , after this the flask should be kept horizontally for proper balancing and aeration. you should check for l-gluatmine in media. L-glutamine decays quicker ( less than 25 days), use long acting l-Glutamine available in the market. L_gluatmine concentrations has greater impact on THP-1 growth. lesser concentrations can even screw up the cell morphology ,as well lead to cell death.also check co2 concentrations , it should always be 5%.
Dear Edwardo, THP-1 has a doubling time of less than 20 hours. The problem lies in the seeding area of cells. THP-1 cells should be grown at a density of 1 million to 2 million/mL .In a 24 well 1 million cells could be easily seeded and grown( no differentiation). In a 250ml culture flask the maximum media should be 50mL and between 20-30 million for seeding , after this the flask should be kept horizontally for proper balancing and aeration. you should check for l-gluatmine in media. L-glutamine decays quicker ( less than 25 days), use long acting l-Glutamine available in the market. L_gluatmine concentrations has greater impact on THP-1 growth. lesser concentrations can even screw up the cell morphology ,as well lead to cell death.also check co2 concentrations , it should always be 5%.
(1) Use this medium: RPMI-1640 (+) L-glutamine + 10% FCS + Penicillin-Streptomycin + Non-essential amino acid + 10 mM HEPES + 50 uM beta-mercaptoethanol because THP-1 cells like this pH environment and beta-mercapoethanol for proliferation If you start from a frozen vial, resuspend cells and maintain them at a high density, give them 3-5 days. Check mycoplasma if necessary.
(2) If (1) is correct, you can see cells round and not in clusters. Pipet up and down for a couple of time before counting single-cell suspensions.
(3) THP-1 cells are a bit tolerant to acidic environment. Usually simply remove 50%-75% old culture and refill new medium to the original volume. Always retain HEPES and maintain the cells at a high density.
(1) Use this medium: RPMI-1640 (+) L-glutamine + 10% FCS + Penicillin-Streptomycin + Non-essential amino acid + 10 mM HEPES + 50 uM beta-mercaptoethanol because THP-1 cells like this pH environment and beta-mercapoethanol for proliferation If you start from a frozen vial, resuspend cells and maintain them at a high density, give them 3-5 days. Check mycoplasma if necessary.
(2) If (1) is correct, you can see cells round and not in clusters. Pipet up and down for a couple of time before counting single-cell suspensions.
(3) THP-1 cells are a bit tolerant to acidic environment. Usually simply remove 50%-75% old culture and refill new medium to the original volume. Always retain HEPES and maintain the cells at a high density.
1) Are you starting your culture from a frozen vial? If so, it can take cells up to one week to recover. By the way I usually seed 2-3x10^5 cells/ml (RPMI-1640 + 10% FBS) and I completely renew the medium every 2 or 3 days. These cells should never exceed 0.8-1x10^6 cells/mL. As far as I know they don't make big clusters, that's why I suggest you to check for any contamination such as mycoplasma. 2) It's possible to count them using the hemacytometer , but you should avoid to pick samples directly from the growing flask. As you know, cells in suspension tend to stay at the bottom of your flask and, even if you resuspend the whole culture, it's nearly impossible to pick an aliquot that reflects the reality of your growing culture, especially if we are talking of 10 to 100 ul. That's why I suggest you to harvest the whole culture, centrifuge, and resuspend using an appropiate volume of fresh media and count one aliquot from this latter one. 3) Personally, I harvest the whole culture, count it if necessary, and seed again approx. 2-3X10^5 cells/ml. Some of my colleagues just remove half of the culture (after resuspending as best as they can) and add the removed amount of media with new supplemented RPMI every 2 or 3 days. I don't like this approach tho, because is error prone, and you risk that your culture exceed 1x10*6 cells/ml. Hope this help.
1) Are you starting your culture from a frozen vial? If so, it can take cells up to one week to recover. By the way I usually seed 2-3x10^5 cells/ml (RPMI-1640 + 10% FBS) and I completely renew the medium every 2 or 3 days. These cells should never exceed 0.8-1x10^6 cells/mL. As far as I know they don't make big clusters, that's why I suggest you to check for any contamination such as mycoplasma. 2) It's possible to count them using the hemacytometer , but you should avoid to pick samples directly from the growing flask. As you know, cells in suspension tend to stay at the bottom of your flask and, even if you resuspend the whole culture, it's nearly impossible to pick an aliquot that reflects the reality of your growing culture, especially if we are talking of 10 to 100 ul. That's why I suggest you to harvest the whole culture, centrifuge, and resuspend using an appropiate volume of fresh media and count one aliquot from this latter one. 3) Personally, I harvest the whole culture, count it if necessary, and seed again approx. 2-3X10^5 cells/ml. Some of my colleagues just remove half of the culture (after resuspending as best as they can) and add the removed amount of media with new supplemented RPMI every 2 or 3 days. I don't like this approach tho, because is error prone, and you risk that your culture exceed 1x10*6 cells/ml. Hope this help.
THP1 usually grow fast unless they are contaminated, differentiated or in bad quality media. From what you are saying they hardly if at all. Options: 1.Dispose and start new another batch -another source of cells. 2 . check versus another cell line as control or change media batch especially serum ( is it heat inactivated ?) 3. check for mycoplasma
THP1 usually grow fast unless they are contaminated, differentiated or in bad quality media. From what you are saying they hardly if at all. Options: 1.Dispose and start new another batch -another source of cells. 2 . check versus another cell line as control or change media batch especially serum ( is it heat inactivated ?) 3. check for mycoplasma
Thawed total 9.2E5 THP-1 cells and mixed into total 15 ml of supplemented RPMI 1640. Actually experimented with variations on serum and other supplements (ferric pyrophosphate and/or beta-mercaptoethanol, the latter being recommended by ATCC), and found that iron-supplemented calf serum was better for initial THP-1 growth than FBS, although it seemed to cause some differentiation, and FBS eventually worked for growth without differentiation (but with a delay of a week after thawing). For growth, finally settled on FBS + supplement of 368 ug/l ferric pyrophosphate, equivalent to 6.6 uM Fe(3+) final concentration in the medium.
ATCC gives a dire warning not to go over 1.0E6 cells/ml, but I found that you can easily exceed this up to 2X without harm. They recommend 50 uM (= 3.9 mg/l = 3.5 ml/l) beta-mercaptoethanol (78.13 g/mole, density 1.114 g/ml), but in practice this seems to be too much and inhibits growth; smaller amounts seem that they may be stimulatory, but I only experimented a few times with this, and any beneficial effect is not huge, and isn't worth the trouble.
Thawed total 9.2E5 THP-1 cells and mixed into total 15 ml of supplemented RPMI 1640. Actually experimented with variations on serum and other supplements (ferric pyrophosphate and/or beta-mercaptoethanol, the latter being recommended by ATCC), and found that iron-supplemented calf serum was better for initial THP-1 growth than FBS, although it seemed to cause some differentiation, and FBS eventually worked for growth without differentiation (but with a delay of a week after thawing). For growth, finally settled on FBS + supplement of 368 ug/l ferric pyrophosphate, equivalent to 6.6 uM Fe(3+) final concentration in the medium.
ATCC gives a dire warning not to go over 1.0E6 cells/ml, but I found that you can easily exceed this up to 2X without harm. They recommend 50 uM (= 3.9 mg/l = 3.5 ml/l) beta-mercaptoethanol (78.13 g/mole, density 1.114 g/ml), but in practice this seems to be too much and inhibits growth; smaller amounts seem that they may be stimulatory, but I only experimented a few times with this, and any beneficial effect is not huge, and isn't worth the trouble.
1) Are you starting your culture from a frozen vial? If so, it can take cells up to one week to recover. By the way I usually seed 2-3x10^5 cells/ml (RPMI-1640 + 10% FBS) and I completely renew the medium every 2 or 3 days. These cells should never exceed 0.8-1x10^6 cells/mL. As far as I know they don't make big clusters, that's why I suggest you to check for any contamination such as mycoplasma. 2) It's possible to count them using the hemacytometer , but you should avoid to pick samples directly from the growing flask. As you know, cells in suspension tend to stay at the bottom of your flask and, even if you resuspend the whole culture, it's nearly impossible to pick an aliquot that reflects the reality of your growing culture, especially if we are talking of 10 to 100 ul. That's why I suggest you to harvest the whole culture, centrifuge, and resuspend using an appropiate volume of fresh media and count one aliquot from this latter one. 3) Personally, I harvest the whole culture, count it if necessary, and seed again approx. 2-3X10^5 cells/ml. Some of my colleagues just remove half of the culture (after resuspending as best as they can) and add the removed amount of media with new supplemented RPMI every 2 or 3 days. I don't like this approach tho, because is error prone, and you risk that your culture exceed 1x10*6 cells/ml. Hope this help.
1) Are you starting your culture from a frozen vial? If so, it can take cells up to one week to recover. By the way I usually seed 2-3x10^5 cells/ml (RPMI-1640 + 10% FBS) and I completely renew the medium every 2 or 3 days. These cells should never exceed 0.8-1x10^6 cells/mL. As far as I know they don't make big clusters, that's why I suggest you to check for any contamination such as mycoplasma. 2) It's possible to count them using the hemacytometer , but you should avoid to pick samples directly from the growing flask. As you know, cells in suspension tend to stay at the bottom of your flask and, even if you resuspend the whole culture, it's nearly impossible to pick an aliquot that reflects the reality of your growing culture, especially if we are talking of 10 to 100 ul. That's why I suggest you to harvest the whole culture, centrifuge, and resuspend using an appropiate volume of fresh media and count one aliquot from this latter one. 3) Personally, I harvest the whole culture, count it if necessary, and seed again approx. 2-3X10^5 cells/ml. Some of my colleagues just remove half of the culture (after resuspending as best as they can) and add the removed amount of media with new supplemented RPMI every 2 or 3 days. I don't like this approach tho, because is error prone, and you risk that your culture exceed 1x10*6 cells/ml. Hope this help.
Hello Eduardo I agree with all the previous answers, however I don't use b-ME and the cells are fine. I also had a problem with culturing THP-1 cells - low viability, an addition of fresh L-glutamine did the trick. Does your medium contain regular glutamine or GlutaMax? In the first case I would suggest adding some fresh glutamine because it tends to degrade fast in the medium. I also never had any problems with counting THP-1 cells, even collecting 10 ul from the flask but I use much smaller volume (10 ml). I always seed them at 0,2 mln/ml and passage at around 0,6-0,7 mln/ml. Most of the time I don't even count then just visually inspect the flask ;) Hope you'll get to a better terms with your cells soon. All the Best Agnieszka
Hello Eduardo I agree with all the previous answers, however I don't use b-ME and the cells are fine. I also had a problem with culturing THP-1 cells - low viability, an addition of fresh L-glutamine did the trick. Does your medium contain regular glutamine or GlutaMax? In the first case I would suggest adding some fresh glutamine because it tends to degrade fast in the medium. I also never had any problems with counting THP-1 cells, even collecting 10 ul from the flask but I use much smaller volume (10 ml). I always seed them at 0,2 mln/ml and passage at around 0,6-0,7 mln/ml. Most of the time I don't even count then just visually inspect the flask ;) Hope you'll get to a better terms with your cells soon. All the Best Agnieszka
(1) Use this medium: RPMI-1640 (+) L-glutamine + 10% FCS + Penicillin-Streptomycin + Non-essential amino acid + 10 mM HEPES + 50 uM beta-mercaptoethanol because THP-1 cells like this pH environment and beta-mercapoethanol for proliferation If you start from a frozen vial, resuspend cells and maintain them at a high density, give them 3-5 days. Check mycoplasma if necessary.
(2) If (1) is correct, you can see cells round and not in clusters. Pipet up and down for a couple of time before counting single-cell suspensions.
(3) THP-1 cells are a bit tolerant to acidic environment. Usually simply remove 50%-75% old culture and refill new medium to the original volume. Always retain HEPES and maintain the cells at a high density.
(1) Use this medium: RPMI-1640 (+) L-glutamine + 10% FCS + Penicillin-Streptomycin + Non-essential amino acid + 10 mM HEPES + 50 uM beta-mercaptoethanol because THP-1 cells like this pH environment and beta-mercapoethanol for proliferation If you start from a frozen vial, resuspend cells and maintain them at a high density, give them 3-5 days. Check mycoplasma if necessary.
(2) If (1) is correct, you can see cells round and not in clusters. Pipet up and down for a couple of time before counting single-cell suspensions.
(3) THP-1 cells are a bit tolerant to acidic environment. Usually simply remove 50%-75% old culture and refill new medium to the original volume. Always retain HEPES and maintain the cells at a high density.
Dear Edwardo,
THP-1 has a doubling time of less than 20 hours. The problem lies in the seeding area of cells. THP-1 cells should be grown at a density of 1 million to 2 million/mL .In a 24 well 1 million cells could be easily seeded and grown( no differentiation). In a 250ml culture flask the maximum media should be 50mL and between 20-30 million for seeding , after this the flask should be kept horizontally for proper balancing and aeration. you should check for l-gluatmine in media. L-glutamine decays quicker ( less than 25 days), use long acting l-Glutamine available in the market. L_gluatmine concentrations has greater impact on THP-1 growth. lesser concentrations can even screw up the cell morphology ,as well lead to cell death.also check co2 concentrations , it should always be 5%.
Dear Edwardo,
THP-1 has a doubling time of less than 20 hours. The problem lies in the seeding area of cells. THP-1 cells should be grown at a density of 1 million to 2 million/mL .In a 24 well 1 million cells could be easily seeded and grown( no differentiation). In a 250ml culture flask the maximum media should be 50mL and between 20-30 million for seeding , after this the flask should be kept horizontally for proper balancing and aeration. you should check for l-gluatmine in media. L-glutamine decays quicker ( less than 25 days), use long acting l-Glutamine available in the market. L_gluatmine concentrations has greater impact on THP-1 growth. lesser concentrations can even screw up the cell morphology ,as well lead to cell death.also check co2 concentrations , it should always be 5%.
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(1) Use this medium:
RPMI-1640 (+) L-glutamine + 10% FCS + Penicillin-Streptomycin + Non-essential amino acid + 10 mM HEPES + 50 uM beta-mercaptoethanol
because THP-1 cells like this pH environment and beta-mercapoethanol for proliferation
If you start from a frozen vial, resuspend cells and maintain them at a high density, give them 3-5 days. Check mycoplasma if necessary.
(2) If (1) is correct, you can see cells round and not in clusters. Pipet up and down for a couple of time before counting single-cell suspensions.
(3) THP-1 cells are a bit tolerant to acidic environment. Usually simply remove 50%-75% old culture and refill new medium to the original volume. Always retain HEPES and maintain the cells at a high density.
Good luck!
(1) Use this medium:
RPMI-1640 (+) L-glutamine + 10% FCS + Penicillin-Streptomycin + Non-essential amino acid + 10 mM HEPES + 50 uM beta-mercaptoethanol
because THP-1 cells like this pH environment and beta-mercapoethanol for proliferation
If you start from a frozen vial, resuspend cells and maintain them at a high density, give them 3-5 days. Check mycoplasma if necessary.
(2) If (1) is correct, you can see cells round and not in clusters. Pipet up and down for a couple of time before counting single-cell suspensions.
(3) THP-1 cells are a bit tolerant to acidic environment. Usually simply remove 50%-75% old culture and refill new medium to the original volume. Always retain HEPES and maintain the cells at a high density.
Good luck!
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Coleen E. P. Thompson Thank you! It is a Gibco 100* solution, and after adding into the medium, working concentration is 1 mM. Hope it helps.
Coleen E. P. Thompson Thank you! It is a Gibco 100* solution, and after adding into the medium, working concentration is 1 mM. Hope it helps.
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1) Are you starting your culture from a frozen vial? If so, it can take cells up to one week to recover. By the way I usually seed 2-3x10^5 cells/ml (RPMI-1640 + 10% FBS) and I completely renew the medium every 2 or 3 days. These cells should never exceed 0.8-1x10^6 cells/mL. As far as I know they don't make big clusters, that's why I suggest you to check for any contamination such as mycoplasma.
2) It's possible to count them using the hemacytometer , but you should avoid to pick samples directly from the growing flask. As you know, cells in suspension tend to stay at the bottom of your flask and, even if you resuspend the whole culture, it's nearly impossible to pick an aliquot that reflects the reality of your growing culture, especially if we are talking of 10 to 100 ul. That's why I suggest you to harvest the whole culture, centrifuge, and resuspend using an appropiate volume of fresh media and count one aliquot from this latter one.
3) Personally, I harvest the whole culture, count it if necessary, and seed again approx. 2-3X10^5 cells/ml. Some of my colleagues just remove half of the culture (after resuspending as best as they can) and add the removed amount of media with new supplemented RPMI every 2 or 3 days. I don't like this approach tho, because is error prone, and you risk that your culture exceed 1x10*6 cells/ml.
Hope this help.
1) Are you starting your culture from a frozen vial? If so, it can take cells up to one week to recover. By the way I usually seed 2-3x10^5 cells/ml (RPMI-1640 + 10% FBS) and I completely renew the medium every 2 or 3 days. These cells should never exceed 0.8-1x10^6 cells/mL. As far as I know they don't make big clusters, that's why I suggest you to check for any contamination such as mycoplasma.
2) It's possible to count them using the hemacytometer , but you should avoid to pick samples directly from the growing flask. As you know, cells in suspension tend to stay at the bottom of your flask and, even if you resuspend the whole culture, it's nearly impossible to pick an aliquot that reflects the reality of your growing culture, especially if we are talking of 10 to 100 ul. That's why I suggest you to harvest the whole culture, centrifuge, and resuspend using an appropiate volume of fresh media and count one aliquot from this latter one.
3) Personally, I harvest the whole culture, count it if necessary, and seed again approx. 2-3X10^5 cells/ml. Some of my colleagues just remove half of the culture (after resuspending as best as they can) and add the removed amount of media with new supplemented RPMI every 2 or 3 days. I don't like this approach tho, because is error prone, and you risk that your culture exceed 1x10*6 cells/ml.
Hope this help.
More
VOTE
THP1 usually grow fast unless they are contaminated, differentiated or in bad quality media. From what you are saying they hardly if at all. Options: 1.Dispose and start new another batch -another source of cells. 2 . check versus another cell line as control or change media batch especially serum ( is it heat inactivated ?) 3. check for mycoplasma
THP1 usually grow fast unless they are contaminated, differentiated or in bad quality media. From what you are saying they hardly if at all. Options: 1.Dispose and start new another batch -another source of cells. 2 . check versus another cell line as control or change media batch especially serum ( is it heat inactivated ?) 3. check for mycoplasma
More
VOTE
Thawed total 9.2E5 THP-1 cells and mixed into total 15 ml of supplemented RPMI 1640. Actually experimented with variations on serum and other supplements (ferric pyrophosphate and/or beta-mercaptoethanol, the latter being recommended by ATCC), and found that iron-supplemented calf serum was better for initial THP-1 growth than FBS, although it seemed to cause some differentiation, and FBS eventually worked for growth without differentiation (but with a delay of a week after thawing). For growth, finally settled on FBS + supplement of 368 ug/l ferric pyrophosphate, equivalent to 6.6 uM Fe(3+) final concentration in the medium.
ATCC gives a dire warning not to go over 1.0E6 cells/ml, but I found that you can easily exceed this up to 2X without harm. They recommend 50 uM (= 3.9 mg/l = 3.5 ml/l) beta-mercaptoethanol (78.13 g/mole, density 1.114 g/ml), but in practice this seems to be too much and inhibits growth; smaller amounts seem that they may be stimulatory, but I only experimented a few times with this, and any beneficial effect is not huge, and isn't worth the trouble.
For differentiating them more fully than what constant phorbol myristate acetate or constant 1,25-dihydroxyvitamin-D[3] will do, see Article The Identification of Markers of Macrophage Differentiation ...
.
Thawed total 9.2E5 THP-1 cells and mixed into total 15 ml of supplemented RPMI 1640. Actually experimented with variations on serum and other supplements (ferric pyrophosphate and/or beta-mercaptoethanol, the latter being recommended by ATCC), and found that iron-supplemented calf serum was better for initial THP-1 growth than FBS, although it seemed to cause some differentiation, and FBS eventually worked for growth without differentiation (but with a delay of a week after thawing). For growth, finally settled on FBS + supplement of 368 ug/l ferric pyrophosphate, equivalent to 6.6 uM Fe(3+) final concentration in the medium.
ATCC gives a dire warning not to go over 1.0E6 cells/ml, but I found that you can easily exceed this up to 2X without harm. They recommend 50 uM (= 3.9 mg/l = 3.5 ml/l) beta-mercaptoethanol (78.13 g/mole, density 1.114 g/ml), but in practice this seems to be too much and inhibits growth; smaller amounts seem that they may be stimulatory, but I only experimented a few times with this, and any beneficial effect is not huge, and isn't worth the trouble.
For differentiating them more fully than what constant phorbol myristate acetate or constant 1,25-dihydroxyvitamin-D[3] will do, see Article The Identification of Markers of Macrophage Differentiation ...
.
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VOTE
1) Are you starting your culture from a frozen vial? If so, it can take cells up to one week to recover. By the way I usually seed 2-3x10^5 cells/ml (RPMI-1640 + 10% FBS) and I completely renew the medium every 2 or 3 days. These cells should never exceed 0.8-1x10^6 cells/mL. As far as I know they don't make big clusters, that's why I suggest you to check for any contamination such as mycoplasma.
2) It's possible to count them using the hemacytometer , but you should avoid to pick samples directly from the growing flask. As you know, cells in suspension tend to stay at the bottom of your flask and, even if you resuspend the whole culture, it's nearly impossible to pick an aliquot that reflects the reality of your growing culture, especially if we are talking of 10 to 100 ul. That's why I suggest you to harvest the whole culture, centrifuge, and resuspend using an appropiate volume of fresh media and count one aliquot from this latter one.
3) Personally, I harvest the whole culture, count it if necessary, and seed again approx. 2-3X10^5 cells/ml. Some of my colleagues just remove half of the culture (after resuspending as best as they can) and add the removed amount of media with new supplemented RPMI every 2 or 3 days. I don't like this approach tho, because is error prone, and you risk that your culture exceed 1x10*6 cells/ml.
Hope this help.
1) Are you starting your culture from a frozen vial? If so, it can take cells up to one week to recover. By the way I usually seed 2-3x10^5 cells/ml (RPMI-1640 + 10% FBS) and I completely renew the medium every 2 or 3 days. These cells should never exceed 0.8-1x10^6 cells/mL. As far as I know they don't make big clusters, that's why I suggest you to check for any contamination such as mycoplasma.
2) It's possible to count them using the hemacytometer , but you should avoid to pick samples directly from the growing flask. As you know, cells in suspension tend to stay at the bottom of your flask and, even if you resuspend the whole culture, it's nearly impossible to pick an aliquot that reflects the reality of your growing culture, especially if we are talking of 10 to 100 ul. That's why I suggest you to harvest the whole culture, centrifuge, and resuspend using an appropiate volume of fresh media and count one aliquot from this latter one.
3) Personally, I harvest the whole culture, count it if necessary, and seed again approx. 2-3X10^5 cells/ml. Some of my colleagues just remove half of the culture (after resuspending as best as they can) and add the removed amount of media with new supplemented RPMI every 2 or 3 days. I don't like this approach tho, because is error prone, and you risk that your culture exceed 1x10*6 cells/ml.
Hope this help.
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VOTE
Hello Eduardo
I agree with all the previous answers, however I don't use b-ME and the cells are fine. I also had a problem with culturing THP-1 cells - low viability, an addition of fresh L-glutamine did the trick. Does your medium contain regular glutamine or GlutaMax? In the first case I would suggest adding some fresh glutamine because it tends to degrade fast in the medium. I also never had any problems with counting THP-1 cells, even collecting 10 ul from the flask but I use much smaller volume (10 ml). I always seed them at 0,2 mln/ml and passage at around 0,6-0,7 mln/ml. Most of the time I don't even count then just visually inspect the flask ;)
Hope you'll get to a better terms with your cells soon.
All the Best
Agnieszka
Hello Eduardo
I agree with all the previous answers, however I don't use b-ME and the cells are fine. I also had a problem with culturing THP-1 cells - low viability, an addition of fresh L-glutamine did the trick. Does your medium contain regular glutamine or GlutaMax? In the first case I would suggest adding some fresh glutamine because it tends to degrade fast in the medium. I also never had any problems with counting THP-1 cells, even collecting 10 ul from the flask but I use much smaller volume (10 ml). I always seed them at 0,2 mln/ml and passage at around 0,6-0,7 mln/ml. Most of the time I don't even count then just visually inspect the flask ;)
Hope you'll get to a better terms with your cells soon.
All the Best
Agnieszka
More
VOTE
(1) Use this medium:
RPMI-1640 (+) L-glutamine + 10% FCS + Penicillin-Streptomycin + Non-essential amino acid + 10 mM HEPES + 50 uM beta-mercaptoethanol
because THP-1 cells like this pH environment and beta-mercapoethanol for proliferation
If you start from a frozen vial, resuspend cells and maintain them at a high density, give them 3-5 days. Check mycoplasma if necessary.
(2) If (1) is correct, you can see cells round and not in clusters. Pipet up and down for a couple of time before counting single-cell suspensions.
(3) THP-1 cells are a bit tolerant to acidic environment. Usually simply remove 50%-75% old culture and refill new medium to the original volume. Always retain HEPES and maintain the cells at a high density.
Good luck!
(1) Use this medium:
RPMI-1640 (+) L-glutamine + 10% FCS + Penicillin-Streptomycin + Non-essential amino acid + 10 mM HEPES + 50 uM beta-mercaptoethanol
because THP-1 cells like this pH environment and beta-mercapoethanol for proliferation
If you start from a frozen vial, resuspend cells and maintain them at a high density, give them 3-5 days. Check mycoplasma if necessary.
(2) If (1) is correct, you can see cells round and not in clusters. Pipet up and down for a couple of time before counting single-cell suspensions.
(3) THP-1 cells are a bit tolerant to acidic environment. Usually simply remove 50%-75% old culture and refill new medium to the original volume. Always retain HEPES and maintain the cells at a high density.
Good luck!
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Bingqian Qu thank you for your medium recommendation. Could you specify the amount of non-essential amino acid?
Bingqian Qu thank you for your medium recommendation. Could you specify the amount of non-essential amino acid?
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