I am trying to study with flow cytometry fern spores. These are big (50 um) heavy cells that sediment while the cytometer is in proces. I need to prepare a medium with higher density to slow down sedimentation. Could I use glycerol? Has anybody used it? What concentration?
Hi Myriam,
there is arather old method using xantham gum to produce a sheath fluid with higher viscosity allowing analysis of heavy particles:
Cytometry. 1989 Nov;10(6):803-6.
Use of xantham gum to suspend large particles during flow cytometric analysis and sorting.
Freyer JP, Fillak D, Jett JH.
I am not quite sure how this would work in modern cytometers but probably worth a try.
Good luck,
Axel
http://www.ncbi.nlm.nih.gov/pubmed/2582972
Viscous solutions may mess with your fluidics.
I would recommend agressive mixing. You may have to acquire for a few seconds, vortex, acquire more. Depending on your architecture you may be able to put a tiny stir bar in the tube and continually mix during aquisition.
I wouldn't try a more viscous sheath, unless you have checked with the manufacturer. I am doing a dynamic monitoring of glucose uptake and in samples with high volumes in order to avoid sedimentation I pause running ang I do a prime. It's not a reccomended trick, but it is easy and you can test it easily
Good luck
Some FACS sorters have agitation devices to prevent sedimentation during prolonged sortings. If you have access to one of those, you could also use it for acquisition.
Thank you all for your suggestions! Due to equipment architecture we cannot stir while acquiring. However it seems the machine itself has an inbuilt stirring system which is not enough. We keep the samples in a sonication bath some minutes and that seems to ameliorate the number of events acquired, but not enough (after some seconds the cells stop passing through). We have also tried increasing detergent (Tween 20) concentration in order to avoid clustering of cells. I will check the protocol with xantham gum.
You may have an issue of macro-aggregates. Do a wet prep and see what you are dealing with.
If your spores are 50 microns, try passing the fluids through a mesh of 100 or 150 microns to strain out macroaggregates.
Another idea is to check the literature and see if there are any pre-treatments that make the spores less sticky.
Dear Mark, yes I think that is the problem and have filtered them through a 50 um filcon twice!. When I observe them under the microscope I do not see clumps, perhaps couples of cells, but it seems they aggregate rapidly, in a few seconds after initiating acquisition. Or could it be other kind of problem?
Searched pollen and aggregation in PubMed and only 56 hits.
Am J Bot. 2014 Sep;101(9):1508-18. doi: 10.3732/ajb.1400312. Epub 2014 Sep 14. suggested "possible selective advantage of the production of aggregated pollen."
This is only one article , but there may be surface properties of the the spores that favor aggregation. The binding of spores to each other could be receptor ligand based or non-specific binding by charge or phobicity. The right pretreatment may help. How aggressive depends on what you are trying to do in the cytometer.
A few quick things to try, incubate in buffer with bovine albumin or nonimmune serum, wash, filter and see.
Other try, trypsinize, wash, filter, and see.
Get fancy - solubilize the spores, run a western blot, and see if spores bind to any bands.
Very Interesting, let us know if you figure this out!
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