Discussion in 'BioMedical ME/CFS Research' started by Andy, Jan 26, 2018.
What a great idea, for interested but non-scientific folk like me. Here's a bit that puzzles me:
Presumably this means all the cells, despite being different types, are much the same size. If they varied much in size then the smaller ones would sometimes go along the tube side by side, and confuse results, especially if different-type small cells went along side by side. Are cells typically similar sizes? Or would they have to only do any single run with similarly sized cells, and tube to match? Or something I'm just missing?
I think the principle is that cells are diluted sufficiently that the chances of getting two passing through the light beam at the same time are very small. A pellet of a million white blood cells fills a bit more than a cubic millimetre. So a sample of 20,000 cells in 0.5ml would make them ~0.005% of the volume if my arithmetic is right. I forget the numbers we used to use but it was somewhere in this ballpark.
The cells are of slightly different sizes. Lymphocytes are generally small and barely more than a nucleus. Monocytes are a bit larger. Blasts and plasma cells can be quite a bit bigger, but they all go down the tube with plenty of room. The type of cell is traditionally identified by a combination of forward and side light scatter, one being a measure of size and the other of granularity. Taken together that tells you pretty definitely whether the cells are neutrophil granulocytes, lymphocytes or monocytes. It may well be that now that it is much easier to use several different fluorescent dyes all at once to label lineage specific surface markers that sorting is now done more or less entirely on fluorescence (CD19 for B cells, CD3 for T cells etc.) but last time I looked at data we always had scatter data as a baseline for picking out the major populations.
Yes, I agree with your arithmetic. That of course is the thing I was missing: that what goes along the tube is mostly fluid, with very spread out cells, relative to their size. That's really interesting, especially the reason for the forward and side light scatter. Thanks.
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