Flow cytometry is a popular technique used to examine the characteristics of cells or particles. It can simultaneously analyse multiple parameters such as relative size, internal complexity, relative fluorescence intensity, etc on the same cell within seconds. Cells are passed through a focused laser beam to produce optical signals including light scattering and fluorescence, which are characteristic to them. There are various technology platforms such as Guava® (Merck Millipore), FACS (Becton Dickinson) and Attune Nxt (Thermo Fisher) that successfully measure physical and chemical properties of cells. Scientists at Aurelia Bioscience are well-experienced at using these platforms for a wide range of cell analysis applications ranging from phenotyping to cell health and viability.
Cell surface markers can be used to define cell subtypes as well as function when labelled with fluorochrome-conjugated antibodies and analysed by flow cytometry. HL-60 cells were stained for CD11b following PMA stimulation to induce differentiation to a macrophage phenotype. Increased CD11b expression was seen with PMA treatment at both 10 and 100nM. In addition, the cells were co-stained with 7-AAD which is taken up by cells when cell membrane integrity is lost indicating decreased cell viability.
Flow cytometry can be used to detect and quantify the level of apoptosis in a population of cells over time. THP-1 cells were stained for Annexin V and 7-AAD following Nocodazole treatment (24hrs). Whilst expression of Annexin V is an early marker of apoptosis, co-staining with 7-AAD results in nuclear staining characteristic of impaired membrane integrity (e.g. necrotic cells).
There are various methods to assess cell proliferation using flow cytometry, such as nucleoside-analog incorporation during DNA synthesis (BrdU and EdU), labelling cell cycle associated proteins (e.g. Ki-67 and PCNA) and dye dilution assays (e.g. CFSE staining). Human T cells were stained with CFSE following IL-2/CD3-CD28 exhaustion. Each peak in the graph represents a cell division and reduced fluorescence intensity observed for each new generation.
In a given population, cells will be distributed among three major phases of cell cycle: G0/G1, S and G2/M phase. At each stage, the amount of DNA present varies. DNA content can be measured using fluorescent DNA stains that exhibit emission signals proportional to DNA mass. Flow cytometric analysis of these stained populations is then used to produce a frequency histogram that reveals te various phases of the cell cycle. THP-1 cells were treated with Nocodazole for 24hrs and then cells were stained with Propidium Iodide. The histogram on the top shows the standard profile for non-synchronised cells and on the bottom shows Nocodazole induced division arrest at G2/M.