02 Apr 2018
Flow cytometry is a laser based, biophysical technology employed in cell counting, sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. When sample solution is injected into a flow cytometer, the particles are randomly distributed. The sample is ordered into a single particle stream then can be interrogated by the machine’s detection system.
After hydrodynamic focusing, each particle passes through one or more beams of light. Light scattering or fluorescence emission (assumed the particle is labeled by a fluorochrome) provides information about the particle’s properties. Fluorescence measurements taken at different wavelengths can provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors or intracellular molecules such as DNA and cytokines. The specificity of detection is controlled by optical filters, which block certain wavelengths while transmitting others.
A flow cytometer is made up of three main systems: fluidics, optics, and electronics.
Any suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis.
Cells from solid tissue must be disaggregated before analysis. The portion of the fluid stream where particles are located is called the sample core.
List mode data are collected on each particle or event. The characteristics or parameters of each event are based on its light scattering and fluorescent properties. The data are collected and stored in the computer. This data can be analyzed to provide information about subpopulations within the sample (Figure 1).
A major application of flow cytometry is to separate cells according to subtype or epitope expression for further biological studies.
Tumor progression is a complex, coordinated and environment-dependent event, and includes cell proliferation, survival, adhesion, invasion and metastasis. Cancer cell metastasis is the main cause of treatment failure, eventually lead to the death .
In mammals, during embryogenesis the cell proliferation leading to the growth of multi adult tissues to fit with various functions. It has been extensively accepted that cancer cells need unlimited replicative effort to generate macroscopic tumors. This property is in clear contrast to the phenomena of the cells in most normal genealogical cells, which are able to breakthrough only a few restricted number of sequential growth and-division cell cycles. This restriction has been related with two a special barriers to proliferation: senescence and crisis. The former is a generally irreversible entrance into a non-proliferative but viable case, but the later includes cell death . Accordingly, when cells are cultured, repeated cell division cycles lead first to creation of senescence and thereafter, for those cells that exceed, by gimmick this barrier, to another phase (crisis phase), in which the vast majority of cells in population die. On rare opportunity, cells arise from a population in crisis and show unlimited replicative force. This transformation has been called immortalization, a recipe that most established cell lines have by their ability to proliferate in culture without proof of either senescence or crisis. Several lines of evidence refers that telomeres protecting a terminals of chromosomes are mainly involved in the ability for unlimited proliferation . The telomeres, form of several tandem hexanucleotide repeats, abbreviate gradually in non-immortalized cells growth in culture, ultimately suffer of losing the ability to save the terminals of chromosomal DNAs from end-to-end fusions; as fusions result from unstable dual-centromere chromosomes whose decision results in an rushes of karyotype that intimidation cell viability. Accordingly, the telomeric DNA length in a cell imposes the number of successive cell generations its lineage can express before telomeres are largely eroded and have then lost their protective functions, stimulating entrance into crisis. Telomerase which is the specialized DNA polymerase that add telomere repeat segments to the terminals of telomeric DNA, is roughly absent in non-immortalized cells but referred at functionally significant levels in the most majority (90%) of voluntarily immortalized cells, involving human cancer cells.
The two proliferation’s barriers, Senescence and crisis/apoptosis are regarded to which have been rationalized as critical anticancer defenses that are hard-wired into our cells, being permeate to hinder the outgrowth of clones of preneoplastic and obviously neoplastic cells. Based on this opinion, most imminent neoplasias consume their grain of replicative doublings and are plugged in their routs by one or the other of these barriers.
During metastasis, primary tumor cells move from the original tumor location, transmitted start [64, 65]. The mechanisms implicit invasion and metastasis were widely an enigma. It was obvious that as carcinomas emerging from epithelial tissues proceed to higher pathological degrees of malignancy, mirrored in native infestation and distant metastasis, the aggregate of cancer cells usually developed modifications in their shape in addition to, in their correlation to other cells and to the extracellular matrix (ECM). The best distinguish variation involved the loss by carcinoma cells of E-cadherin, a cell-to cell key cohesion molecule. By forming adherens sections with neighboring epithelial cells, E-cadherin was helps to aggregate epithelial cell sheets and preserves the cells quietness within these sheets. Expanded expression of E-cadherin was fully determined as a decrease of invasion and metastasis, while its expressions reduction was known to promote these phenotypes. The down regulation and accidental mutational breakdown of E-cadherin are frequently observed in human carcinomas which provided sturdy support for its role as a key inhibitor of this hallmark capability [66, 67]. Moreover, the expression of genes which encoding other cell-to-cell and cell-to-ECM adhesion molecules is obviously altered in some quite aggressiveness carcinomas, with those preferring cytostasis usually being down regulated. On the contrary, adhesion molecules typically aggregated with the cell migrations that take place during embryogenesis and inflammation are frequently upregulated. For instance, N-cadherin, which is usually explained in migrating neurons and mesenchymal cells through organogenesis, is high regulated in several invasive carcinoma cells. After the earning and absence of such cell-cell/matrix correlation proteins, the high regulators of invasion and metastasis were widely unknown or, when dubious, poor in functional validation. The multistage process of invasion and metastasis has been diagramed as a sequence of separated steps, often described the invasion-metastasis cascade. This photography imagine a succession of cell-biologic changes, starting with local invasion, then intravasation via cancer cells into closed blood and lymphatic vessels, passage of cancer cells over the lymphatic and hematogenous systems, then the cancer cells escape from such vessels lumina into the faraway tissues parenchyma (extravasation), then, the last step being termed ‘‘colonization’’ which included formation of small nodules of cancer cells (micrometastases), and eventually the growth of micrometastatic lesions into macroscopic tumors .
Apoptosis, additionally called “programmed cell death” or “cell suicide”, is taken into account a controlled biochemical pathway of celldeath. Technicallyoutlinedby the morphologicaloptions of the dying cell  particularly, by the condensation of the chromatin, typically in the course of fragmentation of the nucleus. Cell death may be a significant part of life in multi cellular organisms, contributing in roles in development, defense, and vital homeostasis. In animals, most necrobiosis is occurring viathe method of programmed cell death, especially on cenecrobiosis behaves as a part of traditionalphysiology. Thecellar membraneis oftencontorted into ‘blebs’,and as well as thecellcouldpercolateseparatemembrane-bound apoptotic bodies. The apoptotic cell and any apoptotic body are rapidly cleared by phagocyte cells, and the cell death couldn’t influence an inflammatory response. Although any cell death within these choices is by definition programmed cell death, we head for typically determine molecular events within the dying cell a show of more classifying the method. Nevertheless, aswe are going tosee, the molecular eventscharacterizationis commonlynot easyas itlooks. Apoptosisismusical team by the action ofa group of proteases in the cell, the caspase (cysteine proteinase with specificity for aspartate residues) . The apoptotic machinery is form of both upstream regulators and downstream effector components . The regulators, in turn state, are classified into two major circuits, one receiving and processing signals which leads to extracellular death-inducing (the extrinsic apoptotic program, including for instance the Fas ligand/Fas receptor), and on the other hand, sensing and merge a different of signals of intracellular origin (the intrinsic program). Every culmination in activation of a normally latent proteases (caspase 8 and 9), which proceed to begin a cascade of proteolysis evolving effector caspase responsible for the application phase of apoptosis, in which the cell is gradually dissociation and then fade out, both by its neighbors and by occupational phagocytic cells. Presently, the essential apoptotic program is more broadly embroiled as a barrier to cancer pathogenesis .
Tumor cells their function through multiple signaling molecules in a time and space dependent manner, which has been widely used as potential therapeutic targets for clinical need. For example, activated Akt1 is associated with changing cell migration and invasion in multi mammalian systems: substantially active Akt1 or its isoforms can strengthen the ability of cancer cells for the invasion in already invasive culture [72, 73] but Akt1 have the opposite effect on less invasive or normal cells . Increased integrinβ1 expression has been shown to associated with augment metastasis in number of cancers [74, 75]. FAK regulates signaling, motility and survival pathways in response to extracellular signals [76, 77], in addition to being a key player in regulating dynamical formation of focal adhesion and cell skeletal reorganization. On another hand, inhibition of FAK activity could largely suppress tumor cell invasion and metastasis. Additionally, during drugs-induced tumor cell apoptosis, PARP is cleaved by caspase into an 89kDa fragment and its ability to respond to DNA strand breaks will be destroyed . In contrast, the discovery of strict target specificities treatments may be useful in eliminating those diseases in the future. Therefore, the search for new agents derived from natural products with a fewer side effects should continue.
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