Introduction to flow with stress on data analysis.
Flow cytometry measures light scattering properties of cells (correlate to cell sizes and cell granularities) and fluorescence emissions of molecules attached to cells (usually conjugates of fluorochrome and antibodies to cell surface proteins). Current cytometers measure quantities of up to 17 surface molecules on each single cell passing through cytometer. Many (thousands to millions) individual cells are measured in a single experiment.
Such experiments provide huge datasets. The analysis of flow cytometry data can be a considerable challenge.
Simple workflow and basic considerations for flow cytometry experiment:
What is sample? Mixture of cells belonging to different groups or populations. Each cell has measurable characteristics. Each population has distributions of these measurable characteristics. Our goal is to separate, quantify and describe populations based on measured parameters. (And on top of this possibly elucidate some type of relations or hierarchy between cell populations in a sample.)
Sample preparation: Cells must be in solution of single cells, so use either plain cell lines, peripheral blood or bone marrow cells or pass solid tissue through a mesh. Cells can be fixed and permeabilized for intracellular labelling.
Cells are labelled with fluorescent dyes, usually conjugated to monoclonal antibodies. Choice of fluorescent dyes
(fluorochromes) depends on the
Cytometer can measure relative size and granularity of cells based on their light scattering properties. It also detects fluorescence of whatever dyes are used. Dyes are usually either conjugated to antibodies to surface (most often) or intracelular proteins or non-Ab dyes. The choice of for example CD surface markers to be measured is critical for what we can say about our sample. Other options are for example measure of endogenous flourescein proteins levels or measurement of DNA content in cells (and subsequently cell cycle analysis).
Labelled cells pass through cytometer in a thin stream of liquid. They are then struck one-by-one by lasers and their
fluorescence is measured. Some cytometers have also sorting ability. In such case different cell populations can be
separated for further experimentation.
Cells are forced to flow in a stream one-by-one by hydrodynamic focusing. It means that sample (itself a liquid cell suspension) is injected into a liquid called sheath fluid, which then forms "liquid tube" around sample. Cells then sequentially interact with light from lasers. The number and type of lasers depend on the instrument. As cells pass through laser light the light is scattered and this scattered light is recorded. Also any fluorescent dye excited by a laser emits light of its characteristic wavelength and this light is also recorded. As emissions of flouorchromes can overlap this overlap must be compensated for before any data analysis.
On cytometers with sorting abilities cell populations can be physically separated based on their measured properties. The sorting is based on deflecting electrically charged droplets containing single cells of interest.
Data from cytometers are usually analysed by examination of graphs. Regions on the graphs correspond to cell populations. By dissecting cell populations, their parameters such as number of cells, average cell size and protein abundance can be assessed.
Regions drawn on plots to dissect cell populations are called gates. There are many types of gates and in a single experiment usually a combination of gates is used to define population of interest. The overall analysis can become very complex when more than two or three fluorescent colours are analysed.
Due to this complexity and to subjectivity of drawing the gates there is considerable effort made to propose some advanced analytical methods for flow cytometry data.
For nice intro see invitrogen's pages.