Flow Cytometry and Cell Sorting
A suspension of cells is hydrodynamically focused in the centre of a fluid stream, where they are passing one by one through a laser beam. Each cell scatters the laser light and fluorescent dyes in the cell or attached to it are excited. This scattered and fluorescent light is picked up by detectors, and, by analysing the brightness at each detector, it is possible to identify various types of cells or to measure functional properties of cells. Flow cytometry has the capability to measure multiple parameters of thousands of cells per second.
Scientists at the HZI are using flow cytometry for:
- Immunophenotyping of surface marker molecules and intracellular proteins, e.g. cytokines or phosphoproteins. We are routinely performing stainings with 10 colours or more to identify and characterise regulatory T cells, B cells, NK cells, or to analyse the cytokine production profile of T cells.
- The analysis of the expression of fluorescent proteins as reporter molecules in tissue culture cells and microorganisms.
- The analysis of physiological properties, i.e. apoptosis, cell cycle or calcium flux.
As an extension of analytical flow cytometry is cell sorting used to separate specific cell types from a heterogeneous population. The sorted cells can be used for cell culture, DNA analysis, RNA analysis, microscopy, protein extraction. Projects involve the sorting of highly pure cell populations, like naïve and regulatory T cells, NK cells and other cell types of the immune system. The sorted cells are used for in-vitro assays and adoptive transfer experiments. A very common application is the purification of rare cells for subsequent gene expression profiling. Another application is the repetitive sorting of fluorescent protein transfected cells to facilitate the generation of stable expressing cell lines.
We currently have several flow cytometers with different performances available:
- BD LSR-II/Fortessa: 5 Laser (360, 405, 488, 561, 640nm), 18 fluorescence colours
- BD LSR-II: 3 Laser (405, 488, 633nm), 11 fluorescence colours
- BD FACSCanto, FACSCalibur and Accuri C6: 2 laser (488, 633nm), 4-6 fluorescence colurs
- BD FACSAria-II: 3 Laser (405, 488, 633nm), 9 fluorescence colours
- BD FACSAria-II SORP: 4 Laser (405, 488, 561, 640nm), 13 fluorescence colours
- BC MoFlo XDP: 4 Laser (405, 488, 561, 640nm), 9 fluorescence colours