Flow cytometry

Flow cytometry is a technique for examining, sorting and enumerating microscopic particles suspended in a stream of fluid. IT is frequently used in counting biological cells, especially blood cells.

In this technique, a beam of monochromatic light (typically a laser beam) is directed onto a narrow stream of fluid containing the particles in question. The particles are then counted and characterized based on their fluorescent or light-scattering properties.

Spot formation

In the aforementioned ELISPOT assays, colored spots are formed within microtiter plate wells. The coloration of these spots depends on the chromogenic substrate used, and their morphology depends on the assay conditions and the type of secretion. These spots can be counted manually or using automated systems (of which I'll say more at a later date).

ELISPOT assays

Previously, I briefly discussed ELISA assays. Out of the ELISA method came the more sophisticated ELISPOT technique, which is a versatile tool for analyzing the substances secreted by peripheral blood and lymphoid cell populations. It is also extremely sensitive, capable of achieving single-cell resolution if properly applied. The ELISPOT technique allows researchers to measure the frequencies (i.e. the relative concentrations) of individual cytokine-, antibody- or granzyme-secreting cells within cultured cell populations.

Here we see how an ELISPOT assay is performed. That link also provides information on reagents for performing a Granzyme B assay.

ELISA assays

The ELISA (Enzyme-Linked Immuno-Sorbent) assay is a biochemical laboratory technique for determining if a certain substance is present in a sample. This is done using antibodies specific to the substance in question. These antibodies are linked to an enzyme which produces a signal.

The sample is first applied to a substrate within an ELISA plate. (This is a type of microtiter plate, designed to work specifically with ELISA assays.) Enzyme-linked antibodies are applied, such that some of them bind to the substance. The unlinked antibodies are then washed away.

Next, a chemical is applied--one which is activated by the enzyme, such that it generates a fluorescent signal. Thus, if the well fluoresces, then the sample is known to contain the substance in question. The intensity of the fluorescences indicates the concentration of this substance.