Hello, my fellow researchers,
I have experience with blood samples flow cytometry and I know both the theoretical and practical stuff behind discretion of singlets (FSC-A/FSC-H) in blood leukocytes,
but recently I got faced with unusual readings in this gate when measuring bronchoalveolar lavage fluids.
The thing is, in absolutely dominating majority of samples, the macrophage population is not following the usual diagonal "line" like in blood.
There are two problems:
The macrophages located at the level of lymphocytes show the same markers as those on the scatter edge area, thus there is no reason to doubt them being macrophages.
The thing I can't wrap my head around is:
If macrophages were as small as lymphocytes (e.g. dying cells), they would be located within the lymphocyte singlets population.
If macrophages were only larger than lymphocytes, but not doublets, they would follow the "singlets diagonal" as they do in blood samples.
Our cytometer, BD FACSAria Fusion, has the laser beam focused into oval shape of rougly 65x9 microns. Could these strange readings be caused by the fact that these macrophages are larger that the beam?
The macrophages in BAL are also extremely granular – could this be the source of the issue?
Why do macrophages form a population in the area one would expect lymphocytes multiplets?
I have attached a FSC-A/FSC-H scattergram of one of my samples to demonstrate the problem.
Thank you for all your insight into this matter.
Hi Ondřej Janča,
To solve problem#1: It happens sometimes (when the cells are too big) that even when you bring the FSC Voltage down close to zero, the cells still show up way higher on the scatter plot (as is the case in the plot you have shared). In such cases, you can switch the neutral density filter (ND Filter) which allows you to put larger scatter events towards lower range of the scatter plot (while still using slightly higher FCS voltages). Luckily, FACSArias have an option to switch ND filter which sits right in front of the obscuration bar (near the Flow cell/Cuvette). I think the default ND filter size on Arias is 1, but please check yours, and try to use a higher one (to block more light). Please note that once you switch this filter, you will need to use higher FSC voltage than you were actually using.
To solver problem#2: The good thing is that you seem to be doing the right thing by trying to locate Macrophages on the scatter plot (back-gating). However, don't you think there are way too many doublets on the plot? Could it be that the debri is showing up in place of Lymphocytes, and the actual Lymphocytes are showing up as what seem to be the doublets in the plot? Wondering, what was the FSC voltage you were using? Could you try tweaking the FSC voltage (even before switching the ND filter) to see if that brings the Macrophages on scale? I am hoping once you fix the FSC voltage and/or ND filter you will start seeing the actual Macrophages which I am suspecting to be mostly off-scale right now (in the plot you shared).
Hope this helps! All the best!!
P.S: Please let me know if that worked :)
Looks like they're activated, and started to attack things, clump and die. Is there viscosity in the solution (genomic DNA)? Look at your protocol and try to find steps causing distress -- like long incubation periods, unusual chemical exposures, mechanical agitation. Anything that could disturb these sensitive cells.
Hello Ondrej,
Don't worry, your macrophages are there. They are not perfect spheres, don't expect them to appear in a 45º line like lymphocytes. If you take for analysis only those plastered against the limit of your plot, you risk taking cells with uncertain characteristics. Macrophages comprise the large population at the center of your plot. Take a look at the attached images. If you need the referred paper I can send it.
Best regards
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