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The HL-60 cell line is an acute myeloid leukemia cell line that is used extensively to study white blood cell differentiation (Gorman and Dabbs, 2009). The HL-60 cell line is a reliable and effective tool in studying the control of cellular differentiation since they can be induced by some chemical agents to differentiate into different phenotypes such as macrophage and granulocyte (Reno and Cannas, 2005). Reno and Cannas (2005) also stated that the HL-60 cell line is important in understanding cellular differentiation as it is the most common cell line used in mammalian cell culture. Freshney (2000) says that the field of cell biology has many wide applications that use the techniques of cell culture like the study of the cell proliferation and differentiation regulators in cytogenetic studies.
The HL-60 cells and Hek293 cells (human embryonic kidney) are grown for days or weeks to reach the optimal number of cells and basic aseptic techniques are essential to protect the cell line from contamination (Gorman and Dabbs, 2009). Cell lines may become confluent so sub-cultured or passage is important to prevent cell death by decreasing the mitotic index (Gorman and Dabbs, 2009). Tenopoulou et al. (2007) says that calcein–AM and propidium iodide (PI) can be used to examine the viability of cells in culture. They also stated that calcein–AM can stain live cells and analyse them because it’s a non-fluorescent lipophelitic ester that can enter the cells plasma membrane and separate by esterases in cytosol to produce calcein. Ozdemir (2007) indicates that PI is used for identifying dead cells however PI can only penetrate a damaged membrane which enables PI to bind to nucleic acid to generate red fluorescence which can be measured using a fluorescent plate reader (Gorman and Dabbs, 2009).
In the process of cell differentiation, cells develop from stem cells to form specialized types of cells and their formation depends on the type of agent the cells are treated with. The HL-60 cell line can be induced to differentiate to different types of cells such as granulocytes, monocytes and macrophages. Sivak and Duuren (1970) say that a tumor promoter like PMA can be used with HL-60 cell lines for more specific differentiation into macrophage-like phenotype however this process needs extra attention to avoid cells death and to find suitable PMA concentration for cells treatment. Jacob, et al. (2002) says that DMSO can also be used for more specific differentiation and reported that after seven to ten days of the treatment of HL-60 cell line by DMSO the cells differentiated to granulocyte-like phenotype. Therefore, the HL-60 cell line is a very important tool to study the cell growth and differentiation by the use of specific agents such as PMA for differentiation to macrophage and DMSO or retinoic acid for the differentiation to granulocyte-like cells (Gruneiro, et al. 1995). Macrophage and granulocyte-like cells are important in the process of phagocytosis and exocytosis, respectively. In phagocytosis macrophage engulf large particles like bacteria, viruses and food and ingest them as can be seen in figure-1 (Zqul, 2004).Omer (1986) says that phagocytosis is a defense mechanism and is involved in tissue repair. The process of phagocytosis starts by non-specific binding and invagination of target particles on the cell surface and ends by the formation of the phagosome (Yin and Stossel, 1982). Phagosome moves and binds with lysosome and after fusion the enzymes inside the lysosome lyses the substances inside it as seen in figure-2 (Yin and Stossel, 1982). In vitro, the microsphere is opsonise with IgG to allow them to be ingested by the HL-60 cells via the Fc receptor presented on the cell surface (Gorman and Dabbs, 2009).
Flow cytometry (FCM) measurements used with phagocytosis provide suitable fast reproducible measures for a single cell in suspension, and also exocytosis can be measured using flow cytometry . In addition to that, FCM has techniques that can be used to assay measure oxidative burst in the cell. Forward scatter (FCM) measurement is concerned with cells size and side scatterwith the granularity of cells (Lehmann, et al. 2000; Gorman and Dabbs, 2009). Exocytosis involves the process of molecules secretion from a cell into extracellular space through vesicles as shown in figure-2 (Gorman and Dabbs, 2009). Exocytosis is used by white blood cells in defense where it release granules with various antimicrobial molecules including superoxide (O2) and hydrogen peroxides (H2O2) (Gorman and Dabbs, 2009). Methods of measuring exocytosis include the use of interference of light and flow cytometry and measuring oxidation of Mitosox red (Llobet, et al. 2003; Gorman and Dabbs, 2009). The last technique depends on the production of free radicals, such as O2- inside the cells and H2O2 in the extracellular space and with the use of dihydrorhodamine (DHR) (Gorman and Dabbs, 2009). Dahlgren and Karlsson (1999) saythat the production of superoxide inside the cells is called respiratory burst which can cause some diseases like the syndrome chronic granulomatous disease (CGD). The main features of this syndrome is the susceptibility to microbial infections associated with the reduction of superoxide consumption.
The culture of animal and human cells and tissues is currently using technology in many different areas from the basic scientific cell and molecular biology to applied disciplines of biotechnology. Presently, the list of different cells and tissues which can be cultured is extensive (Marsh, 2001). It includes cell lines derived from connective tissue, fibroblasts, skeletal tissues, neutral cell, endocrine cells, melanocytes and many different types of tumor. Cell differentiation is defined as a process that responses on signals from extracellular circumstances. The term “cell differentiation” is sometimes restricted to examples of irreversible characterization of cells which have been found in many extreme cases. They might include loss of the genetic material, or dead cells (Marsh, 2001). Cell differentiation can be considered reasonably in prokaryote as well as eukaryote systems. It can also be compared some aspects as diverse as the cell division cycle, metabolic, protein synthesis, hormonal signals and cell specialization. There are five main aspects of cell differentiation such as: signal reception and transformation; selective alterations in the genetic material; differential gene expression; organization of gene expression programs and intercellular coordination of cell differentiation within developmental programs of tissues, organs and organisms (Nover, et al. 1982). Some monocytic cell lines have been used in examining macrophage activity such as U937, THP-1, HL-60, and Mono Mac6 (Sintiprungrat, Singhto, et al., 2010). Amongst those monoctytic cell lines, human promyelocytic leukemia cells (HL-60) are monocytic cell lines that are well known study in the inducible myeloid growth and differentiation of white blood cells. Phorbol 12-myristate 13-acetate (PMA) responses to many features of macrophage differentiation while dimethyl sulfoxide (DMSO) induces for granulocytic differentiation (Rowley, Farley, et al., 1992). According to Chen & Sokoloski, et al. (1998), HL-60 promyelocytic leukemia cells have been applied monocytic/macrophagic differentiation with TPA and Vitamin D3 treatments and DMSO (dimethylsulfoxide) treatment in the role of granulocyte differentiation (Renò and Cannas 2005).
HL-60 cells are bipotent which means they have the capability to grow up a functional granulocyte or a macrophage after treatment. The termination in differentiation of HL-60 can be observed by morphological, biochemical, and immunological changes of properties (Kawaii, Tomono, et al., 1999). In addition, HL-60 can be used for differentiating into a large number of different cell types including granulocytes and macrophages as a basic manipulation by examining the PMA and DMSO concentration (Chaplinski & Sloan, 1984) Also, according to Chaplinski and Sloan (1984), HL 60 can be matured into functional granulocytes and macrophage with responding to some reagent treatments (Figure 1 & 2).
Phagocytosis, antigen presentation, cytokine secretion and chemotaxis are included in biological activities of macrophages. Moreover, the expressions of macrophages associated with several diseases which have prospects in medicine. HL-60 cells can be induced to termination of differentiated granulocytes or monocytes/macrophages in responding to variable inducers. It is found that tescalcin is a 25-kDa EF-hand Ca2+ binding protein can unregulate in human leukemia HL-60 cells. It has been induced to differentiate along the granulocytic lineage. Whilst, macrophage-like differentiation of HL-60 cells the expression of tescalcin is downregulated (Levayand & Slepak, 2010). Furthermore, the use of flow cytometer for analyzing of individual particles is significantly important for researcher in cell differentiation. The individual particles can be analyzed by flow cytometer from blood, bacteria, sperm and plankton in cells and tissues. Therefore, a flow cytometer can be involved an instrument that illuminate cells when they flow individually in front of a light source. Then, it detects and correlates the signals from those cells that result from the illumination (Hawley & Hawley 2004).
The aims and approach of the project outline basically the applications of HL-60 cell line in cell differentiation by using DSMO and PMA to induce and differentiate HL-60 into phagocytosis and exocytosis. The studies of phagocytosis and exocytosis processes are clearly explained following results and each method which is used during experiments. Using PMA and DMSO treatmenst to observe the phenotype of macrophage and granulocyte- like cells are focused. Moreover, the use of fluorescence plate reader and flow cytometer in optimizing the optimum concentration of PMA and DMSO is significantly determined. It also aims to familiarise with technique which using in science such as fluorescence microsphere measurements, cell phenotyping and culture involved cell analysis in cell differentiation (Gorman & Pour, 2010).
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