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Cytoflex Flowcytometer, clotting issues and cleaning procedures?
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Nadide Torun
Cytoflex Flowcytometer, clotting issues and cleaning procedures?
Dear Gary Lee Gilmore , Michael G. Weller and Annemieke Ten Bokum . Thank you very much for your response. We have now decided on a more rigourous maintenenance protocol with increased frequency of washing and regular deep cleaning over night. For now, this seems to work well. Thansk again!
Dear Gary Lee Gilmore , Michael G. Weller and Annemieke Ten Bokum . Thank you very much for your response. We have now decided on a more rigourous maintenenance protocol with increased frequency of washing and regular deep cleaning over night. For now, this seems to work well. Thansk again!
We have been having similar issues with the Cytoflex, with a strange looking population of cells appearing on some but not all samples, cleaning does seem to improve this, has anyone else noticed this ?
We have been having similar issues with the Cytoflex, with a strange looking population of cells appearing on some but not all samples, cleaning does seem to improve this, has anyone else noticed this ?
I have run similar types of cells on my flow cytometers [both Beckman-Coulter and Becton-Dickinson], so I have some experience with this issue, though I have not had experience with an automated sample system.
I would say there could be two different issues you may be dealing with: clotting and clumping.
Dead and dying cells can release their DNA, which clumps cells together. This can be severe if you are analyzing samples that have been cryopreserved. You can circumvent this if you use DNAse in your sample buffer, or use Tyto Running Buffer from Miltenyi Biotec. It is expensive, but well worth the cost if your samples are precious. Alternately, if you cannot use the Tyto Running buffer in your assays, you could run it at the start of your cleaning procedure to clean up your machine.
Clotting is activation of the clotting cascade, and is dependent on the presence of divalent cations such as Mg+2 and Ca+2 and fibrinogen, a major component of serum. This would be particularly problematic in your platelet activation studies. For clotting to be an issue, you would need to be using serum - as opposed to BSA or HSA - as your non-specific protein component of your sample buffer your cells are in. If you are using serum, I would switch to BSA or HSA and see if that helps. If not, I would run a non-specific protease solution - such as pronase to degrade the clots at the start of the cleaning procedure.
I have run similar types of cells on my flow cytometers [both Beckman-Coulter and Becton-Dickinson], so I have some experience with this issue, though I have not had experience with an automated sample system.
I would say there could be two different issues you may be dealing with: clotting and clumping.
Dead and dying cells can release their DNA, which clumps cells together. This can be severe if you are analyzing samples that have been cryopreserved. You can circumvent this if you use DNAse in your sample buffer, or use Tyto Running Buffer from Miltenyi Biotec. It is expensive, but well worth the cost if your samples are precious. Alternately, if you cannot use the Tyto Running buffer in your assays, you could run it at the start of your cleaning procedure to clean up your machine.
Clotting is activation of the clotting cascade, and is dependent on the presence of divalent cations such as Mg+2 and Ca+2 and fibrinogen, a major component of serum. This would be particularly problematic in your platelet activation studies. For clotting to be an issue, you would need to be using serum - as opposed to BSA or HSA - as your non-specific protein component of your sample buffer your cells are in. If you are using serum, I would switch to BSA or HSA and see if that helps. If not, I would run a non-specific protease solution - such as pronase to degrade the clots at the start of the cleaning procedure.
I would highly recommend to do the hypochlorite bleach cleaning followed by cleaning solution and ddH2O which you use for startup, also at the end of the run. I don't see the point of the trypsin treatment. I don't know exactly the composition of the Cytoflex sheath fluid, but I suspect it is PBS with EDTA, which should reduce clumping. You can do a monthly sample line clean as recommended by the manufacturer. Usually this means filling the system with hypochlorite (max. 3%) and letting it sit overnight. This is a treatment recommended for removing biofilms and clots/clumps. Since your machine sounds like it is used for many different purposes, I would insist that all samples are fixed before they are run, and also filtered over cell strainers at several steps during their preparation. This is especially important if you are doing cell sorting by flow cytometry, where a blockage in the system could cause you to lose a precious experimental sample.
I would highly recommend to do the hypochlorite bleach cleaning followed by cleaning solution and ddH2O which you use for startup, also at the end of the run. I don't see the point of the trypsin treatment. I don't know exactly the composition of the Cytoflex sheath fluid, but I suspect it is PBS with EDTA, which should reduce clumping. You can do a monthly sample line clean as recommended by the manufacturer. Usually this means filling the system with hypochlorite (max. 3%) and letting it sit overnight. This is a treatment recommended for removing biofilms and clots/clumps. Since your machine sounds like it is used for many different purposes, I would insist that all samples are fixed before they are run, and also filtered over cell strainers at several steps during their preparation. This is especially important if you are doing cell sorting by flow cytometry, where a blockage in the system could cause you to lose a precious experimental sample.
I think that there might be a third problem: Biofilms. In all systems with aqueous buffers, which are not 100% sterile, biofilms will grow. They are extremely persistent. Short cleaning cycles might be sufficient to avoid them, but they will not remove them properly.
I think that there might be a third problem: Biofilms. In all systems with aqueous buffers, which are not 100% sterile, biofilms will grow. They are extremely persistent. Short cleaning cycles might be sufficient to avoid them, but they will not remove them properly.
Dear Gary Lee Gilmore , Michael G. Weller and Annemieke Ten Bokum . Thank you very much for your response. We have now decided on a more rigourous maintenenance protocol with increased frequency of washing and regular deep cleaning over night. For now, this seems to work well.
Thansk again!
Dear Gary Lee Gilmore , Michael G. Weller and Annemieke Ten Bokum . Thank you very much for your response. We have now decided on a more rigourous maintenenance protocol with increased frequency of washing and regular deep cleaning over night. For now, this seems to work well.
Thansk again!
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We have been having similar issues with the Cytoflex, with a strange looking population of cells appearing on some but not all samples, cleaning does seem to improve this, has anyone else noticed this ?
We have been having similar issues with the Cytoflex, with a strange looking population of cells appearing on some but not all samples, cleaning does seem to improve this, has anyone else noticed this ?
More
VOTE
I have run similar types of cells on my flow cytometers [both Beckman-Coulter and Becton-Dickinson], so I have some experience with this issue, though I have not had experience with an automated sample system.
I would say there could be two different issues you may be dealing with: clotting and clumping.
Dead and dying cells can release their DNA, which clumps cells together. This can be severe if you are analyzing samples that have been cryopreserved. You can circumvent this if you use DNAse in your sample buffer, or use Tyto Running Buffer from Miltenyi Biotec. It is expensive, but well worth the cost if your samples are precious. Alternately, if you cannot use the Tyto Running buffer in your assays, you could run it at the start of your cleaning procedure to clean up your machine.
Clotting is activation of the clotting cascade, and is dependent on the presence of divalent cations such as Mg+2 and Ca+2 and fibrinogen, a major component of serum. This would be particularly problematic in your platelet activation studies. For clotting to be an issue, you would need to be using serum - as opposed to BSA or HSA - as your non-specific protein component of your sample buffer your cells are in. If you are using serum, I would switch to BSA or HSA and see if that helps. If not, I would run a non-specific protease solution - such as pronase to degrade the clots at the start of the cleaning procedure.
I have run similar types of cells on my flow cytometers [both Beckman-Coulter and Becton-Dickinson], so I have some experience with this issue, though I have not had experience with an automated sample system.
I would say there could be two different issues you may be dealing with: clotting and clumping.
Dead and dying cells can release their DNA, which clumps cells together. This can be severe if you are analyzing samples that have been cryopreserved. You can circumvent this if you use DNAse in your sample buffer, or use Tyto Running Buffer from Miltenyi Biotec. It is expensive, but well worth the cost if your samples are precious. Alternately, if you cannot use the Tyto Running buffer in your assays, you could run it at the start of your cleaning procedure to clean up your machine.
Clotting is activation of the clotting cascade, and is dependent on the presence of divalent cations such as Mg+2 and Ca+2 and fibrinogen, a major component of serum. This would be particularly problematic in your platelet activation studies. For clotting to be an issue, you would need to be using serum - as opposed to BSA or HSA - as your non-specific protein component of your sample buffer your cells are in. If you are using serum, I would switch to BSA or HSA and see if that helps. If not, I would run a non-specific protease solution - such as pronase to degrade the clots at the start of the cleaning procedure.
More
VOTE
I would highly recommend to do the hypochlorite bleach cleaning followed by cleaning solution and ddH2O which you use for startup, also at the end of the run. I don't see the point of the trypsin treatment. I don't know exactly the composition of the Cytoflex sheath fluid, but I suspect it is PBS with EDTA, which should reduce clumping. You can do a monthly sample line clean as recommended by the manufacturer. Usually this means filling the system with hypochlorite (max. 3%) and letting it sit overnight. This is a treatment recommended for removing biofilms and clots/clumps.
Since your machine sounds like it is used for many different purposes, I would insist that all samples are fixed before they are run, and also filtered over cell strainers at several steps during their preparation. This is especially important if you are doing cell sorting by flow cytometry, where a blockage in the system could cause you to lose a precious experimental sample.
I would highly recommend to do the hypochlorite bleach cleaning followed by cleaning solution and ddH2O which you use for startup, also at the end of the run. I don't see the point of the trypsin treatment. I don't know exactly the composition of the Cytoflex sheath fluid, but I suspect it is PBS with EDTA, which should reduce clumping. You can do a monthly sample line clean as recommended by the manufacturer. Usually this means filling the system with hypochlorite (max. 3%) and letting it sit overnight. This is a treatment recommended for removing biofilms and clots/clumps.
Since your machine sounds like it is used for many different purposes, I would insist that all samples are fixed before they are run, and also filtered over cell strainers at several steps during their preparation. This is especially important if you are doing cell sorting by flow cytometry, where a blockage in the system could cause you to lose a precious experimental sample.
More
VOTE
I think that there might be a third problem: Biofilms. In all systems with aqueous buffers, which are not 100% sterile, biofilms will grow. They are extremely persistent. Short cleaning cycles might be sufficient to avoid them, but they will not remove them properly.
I think that there might be a third problem: Biofilms. In all systems with aqueous buffers, which are not 100% sterile, biofilms will grow. They are extremely persistent. Short cleaning cycles might be sufficient to avoid them, but they will not remove them properly.
More
VOTE