We have been having clotting issues with our Cytoflex S from Beckman Coulter. Back in December 2020, we had to have the tubing system and sample needles replaced, but we are beginning to observe clotting issues again.
Our current cleaning procedure is to run 5 wells of 2% hypochlorite (60 sec, 60uL/min) followed by the built in “Daily Clean Program” (3 wells of cleaning solution and 5 wells of deionized water) prior to analyzing samples. After analysis, we run 5 wells of Trypsin (60 sec, 60uL/min) followed by the “Daily Clean Program”.
Our Cytoflex is used for a variety of cells, incl. tumor cell lines, PBMCs, monocyte-derived macrophages, and platelet activation studies in whole blood.
Does anyone have experience using these cell types and which cleaning procedures have you implemented to maintain your flow cytometers?
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 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.
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