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Now, we are moving from multiplex type assays to single-cell assays.
We can look here for intracellular cytokine expression
or for the expression of cytokines that are sitting on the surface of cells.
Here, we are looking at human T lymphocytes which are briefly incubated
with an antigen or peptide or activating agents, such as PMA or ionomycin.
If they are permeabilized
and co-incubated with label antibody, fluorescence-labeled antibody,
which is specific for cytokine one, for example and
another antibody specific for cytokine number 2,
which is labeled with Phycoerythrin.
Nuclei are stained with a reagent called DAPI,
shown here in blue and immunofluorescence images,
which can be acquired in an immunofluorescence microscope or by flow cytometry.
What you then see in the images shows the expression of cytokine 1 and
cytokine 2 in the same cells.
Then, you can merge the microscope images and
this confirms co-expression of both cytokines in
the same cell because here the red and the green give you a yellow color and you
can clearly see that the same cytokine RB present are being made in the same cells.
These similar single-cell assays can actually be done without permeabilization of cells
because you can add detection.
Antibody to cells and
if the cytokine is sitting on the surface of the cell,
or if it's sitting bound to the receptor that is present on the surface of the cell,
your detection-labeled antibody will detect the cytokine sitting on a surface,
and that doesn't require permeabilization of the cells.
