After separation from whole blood with a density-gradient medium such as Polymorphprep, two fractions are obtained: PBMCs and PMNs. We know that PBMCs contain Lymphocytes and Monocytes. Is it possible to differentiate these live Lymphocytes and Monocytes based on size only ?
It is known that lymphocytes are generally smaller than Monocytes. However, I have come across resources that talk of the existence of smaller-sized Monocytes (see attached article). So, I would like to know if size is a robust-enough criterion.
It would be very helpful to me if you could share your experience.
As in with what? Flow or just under the microscope?
If you are talking about unstimulated PBMCs... it is possible to tell the difference between monocytes and lymphocytes. But it's more than just size, they are usually differentiated by the size and shape of the nucleus.
For flow... we use SSC and FSC... and yes you can tell the difference between the populations without a marker. With back gating on CD3, I have never had a monocyte appear in the lymphocyte gate in SSC and FSC.
If you are talking about stimulated PBMCs... size may change depending on the strength of the stimulant... and monocytes will become macrophages in plate cultures.
Shen-An Hwang, yes the PBMCs are unstimulated. I'm doing live nuclear staining and image using Brightfield and fluorescence modalities to look at both the cell size and nucleus respectively. Recently, I've started doing high-magnification imaging and things are looking promising.
I am also fluorescently tagging live PBMCs and plating them to confirm monocytes.
Definitely yes, In flowcytometry or in Microscopy you can differentiate lymphocytes and monocytes.
Or if you want evident marker, just add CD45, it will clear all the debris and differentiate both the population.