We frequently experience a strange peak in the forward scatter data acquired with a Partec CyFlow Space cytometer and we have no idea as to what the cause for that might be. We tried various samples, even prefiltered MQ water with various sheath fluids, but the outcome remained the same. Running the cleaning fluid provided by the manufacturer seems to solve the problem, but as soon as anything else enters the flow cell, the peak reappears. This is particularly troublesome in dot plots, e.g. a fluorescence channel vs forward scatter, where the peak distorts the image of a population. Has anyone experienced similar problems or has any idea for a solution? Could this be an instrumentation fault or a physical phenomenon I am not familiar with? Any help would be highly appreciated. I attached a histogram and a dot plot for demonstration.
The peak would seem an artifact due to a threshold value set in a different channel. Have you tried to set the threshold in the red channel? Have you tried to adjust the voltages of the FSC detector? I see that the FSC peak value is rather low and close to what I could expect as instrumental noise. I do not have much experience with your machine, but that's my experience...hope this could be of help.
Hello, Partec Instruments have a high sensitivity FSC parameter, keeping in mind that you are working in Log scale, there is an effective distance between your population and the beginning of the scale. I assume that your threshold is trigger in other parameter, although in Dot plot this is not clear. I would try to acquire in linear scale and see what happen with the smaller peak, also I will test with beads of different sizes to characterize your noise. If it is something related to FSC PMT, changing the voltage you will be able to detect if something goes wrong, hope this could help.
Hello, this curious effect is almost certainly related to dirt in your tube system (sheeth fluid tube) or in/on your flow cuvette. You should contact Partec service for help.
I agree with all the answers above. We had a similar problem and it was caused by dirty lines coming from the sheath tank. A good cleaning solution is 50% DMSO in water. Also, depending on what you measure, a linear FSC setting might push the signal below you threshold.
Hello,Partec PMTs are so sensitive and amplification like Logarithmic scale can be the cause of peak.
In my opinion, I'd also tried to clean the fluidics, we use chlorine solutions. It always helped us to eliminate undesirable noise.
I agree with the previous answers, you have to clean the aparatus, the tubes, and specially the tip. It is better to sonicate the tip and to clean with chlorine solution and a lot of distilled water,. Good luck
Partec instrument is an analyzer and it has no tip. If you sonicate the flow cell you will destroy it. I suppose that Karoly has the instrument in good conditions of cleaning and maintenance and what happen is something related to the way the samples are acquired. To change the amplification and the threshold can help to elucidate the solution, good luck.
On which channel did you set the trigger? Obviously not on FSC. Why not? Does it look different if you set the trigger on FSC?
I share Stefano's idea : due to the small amounts of particles on the left side of the density plot, I do believe it is an artefact related to the threshold value. Just increase it and see if it does change anything. Did you look at the side scatter signal? Much better when dealing with small particles (bacteria for instance).
Good luck.
I do not use this same instrument, but I do not think this is an instrument problem.
We typically do a histogram of Side Scatter (log scale) versus Forward Scatter (linear scale) and we see this same type of pattern. We gate on the main population of cells in this plot and all analysis. If you acquire this (Forward Scatter) in a linear scale - or convert the data to a linear scale the population will broaden (left to right). The vertical population is debris. Then you need to set the Threshold value which will ignore all events below the Threshold you set. You can test that this is a debris problem if you can clean up the cells you use - or use beads - before you run them on the FACS. The vertical population should be reduced significantly. Also, if you take a good / fresh healthy population of cells - do not treat them or label them - and wash them. Run these on the FACS and the vertical population should look better. The other test would be to take a sample of those same fresh healthy cells and heat them / protease treat them ... something to damage them and then run these. This should produce a more prominent vertical population. You might also want to check out your labeling protocol to be certain you are not damaging the cells during your assay. Some cell types seem to be more fragile than others. You can check this by saving samples at each step of your assay and then examining them by Forward versus Side Scatter.
Hi, I agree with some colleagues about "dirt in your tube", but I would check also the presence of bubbles in your flow chamber.
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