Flow cytometry is a method
for analyzing particles.
These could be cells or
other types of particles.
In this example, we have lymphocytes incubated
with a fluorescently tagged monoclonal antibody.
Perhaps we want to enumerate the number of CD4+
cells and the number of CD8+ cells in this patient.
A sheath fluid containing the antibody labeled cells passes
through a small narrow tube where
there is just one cell per drop.
And laser light is shone onto these cells,
passing through one by one down this small tube.
One can measure forward light scatter from the laser
light which gives a indication of the size of the cell.
One can also measure 90 degree light scatter
which indicates the granularity of the cell.
Red and green fluorescence detectors will detect monoclonal
antibodies that are binding to either CD4 or CD8.
So one monoclonal antibody against CD4 might
be labeled with a dye that emits green
fluorescence, and the other antibody against
CD8 with a dye that detects red fluorescence.
The forward scatter and side scatter
allows detection of different cell types.
And we can see here an example of the type of plot that
the computer linked to the flow cytometer produces.
You can see the side scatter on the Y-axis
and the forward scatter on the X-axis.
And one is able to differentiate neutrophils
from monocytes, from lymphocytes.
Using monoclonal antibodies, one is
able to detect different populations
of cells as already mentioned, for example, CD4 and CD8 T-cells.
Here we can see the CD4+ T-cells that have been stained
with a monoclonal antibody coupled to the diphycoerythrin.
And the CD8+ cells detected by a monoclonal antibody
coupled to the difluorescein isothiocyanate.
The cells that are not staining are mainly B-cells and NK cells
because these cell types lack expression of both CD4 and CD8.
Here we see another use of this technology,
detecting the double positive thymocytes.
Thymocytes are early T-cells that
are developing in the thymus.
This is the only location that you ever find T-cells expressing
both CD4 and CD8 because once they leave the thymus, they
switch off expression of one of these two genes to become either
CD4 single positive T-cells or CD8 single positive T-cells.
But here we can see very clearly on this flow cytometry
plot that there are a number of cells that are
staining for both CD4 and CD8; very typical of
developing thymocytes, double positive, CD4+ CD8+ cells.
Flow cytometry can also be used for the detection of specific
T-cells using a technique that employs peptide MHC tetramers.
Fluorescently labeled tetramers of four identical
HLA molecules are loaded with particular peptides.
In this example, a peptide derived
from the Epstein-Barr virus.
This allows enumeration of
antigen specific T-lymphocytes.
The fluorescence-activated cell
sorter is a modified version of the
flow cytometer that allows the separation
of different cell populations.
Here we have a mixed population of cells
labeled with different fluorescent antibodies.
The mixture of cells is passed through the flow cytometer
and laser light is shone onto the individual cells.
A dichroic filter allows the detection of forward
scatter and also of fluorescence in different
fluorescent channels depending on which particular
fluorescent label is tagging the monoclonal antibody.
Also a side scatter detector can
be used for analysis of the cells.
The computer that analyses all of the
inputs from these different detectors and
will provide a display on the computer,
whereby one can see the different cells.
So here you can see some cells are double positive,
in other words are tagged with both antibodies.
Some are single positive and
some are completely negative.
So this is the kind of information one
would get from a standard flow cytometer.
But in the fluorescence-activated cell sorter, the cells
as they pass through in a single stream can be deflected
in different directions depending on a charge that is
put on the droplet containing the individual cell.
And as we can see here, the cells can
be separated into these four different
populations - ones that are positive
just for one antibody binding or the
other antibody binding, in other words
single positive cells, the double positive
cells and the double negative cells,
separated into four different tubes.
One can then go on and analyze these
different populations, in a variety of ways.