In the nitroblue tetrazolium test
(NBT), the pale yellow color of
NBT changes to dark blue in neutrophils stimulated with phorbol
myristate acetate (PMA) which is used to induce a reactive
oxygen burst in these cells, producing reactive oxygen species.
So a burst of respiration that normally occurs
following phagocytosis of microorganisms.
In a normal donor, we can see this dark blue precipitate that
occurs over the neutrophils indicating that they have indeed
mounted a respiratory burst producing reative oxygen species
following this stimulation, artificial stimulation with PMA.
In a patient with chronic granulomatous
disease, which is an immunodeficiency in which
patients are unable to produce reactive oxygen
species, there is no reduction of the NBT.
And therefore these dark patches
do not appear over the neutrophils.
The production of reactive oxygen species can
also be measured using dihydrorhodamine 123.
The T-cell receptor excision circle or TREC assay is
used to analyze the output of T-cells from the thymus.
TRECs are circular DNA molecules that are produced
during T-cell receptor gene recombination.
They can be measured using the
polymerase chain reaction, PCR.
And this assay is used to quantify recent thymic
emigrants as a measure of T-cell output from the thymus.
So here we have an example of the
T-cell receptor α-chain gene locus.
And as T-cells develop within the thymus they
will recombine their T-cell receptor genes.
And in the case of the T-cell receptor α-chain,
will place a Vα gene segment adjacent to a Jα gene segment.
And the intervening bit of DNA forms a circle, the
T-cell receptor excision circle that we can see here.
And the TREC assay measures the generation of these circles
to give an indication of T-cell output from the thymus.
Lymphocyte responses can be measured
via the proliferation assay.
In the case of T-cells, the substance
phytohemagglutinin (PHA) acts as a mitogen.
A mitogen is a substance that generates
mitosis, causes cell division.
So these cells will be polyclonally activated,
that means they become activated irrespective of
their particular antigen specificity and they will
start to divide over and over and over again.
By adding the isotope of thymidine, thritiated thymidine,
this will become incorporated into the cells as they divide.
It’ll be incorporated into
their newly produced DNA.
And one can measure the
radioactivity in the 3H-thymidine.
The ELISPOT assay which stands for ELISA SPOT
assay is used to measure cytokine release
or indeed can be used to measure the protein-- the level
of protein released from different cell types.
So it could be a hormone for example.
But here we’re looking
at a cytokine release.
Peripheral blood cells which
consists of lymphocytes and monocytes
are added to a dish that is coated
with a anti-cytokine antibody.
These cells can release cytokines and
particularly the lymphocytes in response
to incubation with soluble antigen, will
be stimulated to release cytokines.
After culturing the cells with antigen for two days, we can see
in this particular assay that Th2 cells are secreting IL-4.
Interleukin-4 is a characteristic
cytokine of Th2 cells.
And they are secreting this in
response to the soluble test antigen.
The IL-4 released from these cells is captured
by the antibody that is coating the plate.
So following the incubation, the release
of the cytokine, the cytokine being
bound by the antibody, captured by the
antibody, the cells are then washed away.
And the cytokine is revealed by a second antibody, which is a
labeled antibody against another epitope on the same cytokine.
A chemical reaction gives
spots as we can see here.
And you can see on the left hand side, a example
of cells that are not secreting interleukin-4.
And on the right, some spots showing that
some of the cells have secreted interleukin-4.
And one is able to enumerate these, if you know how
many cells you added to the assay, then you can get an
approximation of the percentage of cells that are
secreting this particular cytokine, the ELISPOT assay.
It’s also possible to measure
cytotoxic T-cell activity.
In this assay, cells are
labeled with chromium-51.
And these are mixed with effector cells
that are able to mediate cytotoxicity.
So these could be natural killer cells for
example, of the innate response, or they
could as shown here be the cytotoxic
T-lymphocytes of the adaptive immune response.
If the labeled cells are capable of being killed
by the cytotoxic T-lymphocytes, there will
be release of radioactivity into the culture
fluid from the cells as they become killed.
So as the target cells are killed,
they release the radioactive isotope.
And this, the amount released will
be proportional to target cell lysis.