International Research Journal of Education and Innovation (IRJEI)
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August 2015
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Isolation, Culture Procedures and Microscopy of the Mouse Peritoneal
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Cells for Assaying Phagocytosis: An Undergraduate Laboratory
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Experiment
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Gaber Ramadan1,†, Aysha Al Neyadi1,†, Ahmed Mashli1,RabahIratni1,*and Synan AbuQamar1,*
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Department of Biology, United Arab Emirates University, PO Box 15551, Al-Ain, United Arab
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Emirates.
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These authors contributed equally to this study.
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*Corresponding author:Synan AbuQamar (Email:sabuaqamar@uaeu.ac.ae; Telephone: +971 (3)
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713-6733); and RabahIratni(Email:R_iratni@uaeu.ac.ae; Telephone: +971 (3) 713-6526).
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Abstract
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To investigate phagocytosis, a class of undergraduate students isolated, cultured and fixed the mouse
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peritoneal cells. We concluded that the mouse immune system has high efficiency to eliminate
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pathogens. Educational research has shown many advantages of using “hands-on” experiments
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which will help students carry out different experimental techniques and ultimately gain laboratory
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research skills. Skills that include preparing tissue culture media, handling animals and cell culture
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techniques, staining, and utilizing microscopes, will develop the knowledge needed to work in a
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laboratory, to produce a plan to undertake and test scientific hypotheses, and to emphasize future
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employability skills and attitudes.
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Key words:Active-learning strategies, peritoneal cells,phagocytosis, microscopy, mouse.
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Introduction
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Many science education reports have shown that traditional lectures accompanied with “ready
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recipe” labs do not serve the needs of the student during his/her educational development (Smith et
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al., 2005; Wood, 2003). Therefore, active-learning strategies, also known as seeking new
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information, are very important to teach Biology as a discipline to undergraduate students. These
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strategies can mainly be present in the laboratory work in which the students are highly engaged in
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practical experiments, rather than having an instructor as the center of the class to run the “show”.
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Such laboratory practices or classes provide great opportunities to students to handle different
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“wethand” procedures. This increases the students understanding and makes them rely on the
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evidence rather than theoretical knowledge. To illustrate, it clarifies/explains an observed event
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through connecting their theoretical knowledge with the practical skills (Bendassolli,2013).
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Furthermore, laboratory work increases the student's awareness about the research methodology.
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Altogether, this may help the students in learning scientific concepts through scientific methods in
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order to understand the nature of science. The effective laboratory handling helps the student
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investigate and understand various scientific concepts. The goal of having a laboratory work is to
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incorporate active learning strategies and develop students with essential skills for industry and
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research laboratories. Nowadays, the needs of research have undoubtedlybecome much more
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specialized as biological knowledge has expanded (Cardak et al.,2007; Ottander & Grelsson 2006;
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Tan, 2008).
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Theanimal immune system is made up of a network of cells, tissues and organsthat work
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togetherto protect the body and eliminate the infections. This function is carried by the action of
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several immune cells, which originate from pluripotent hematopoietic stem cells found in the bone
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marrow (Roittet al., 1998). The immune cells are mainly leukocytes which are mainly classified into
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granulocytes and agranulocytes. The granulocytes are composed of basophils, eosinophils, and
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neutrophils, while the agranulocytes include monocytes, natural killer cells and lymphocytes
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(Burkittet al., 1993). Neutrophils, eosinophils, basophils, and monocytes are the main blood
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phagocytic cells. Within few hours after infection, monocytes can move quicklyto the sites of
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infection inthe tissues in order to allow them to divide/differentiate into macrophagesto elicit an
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immune response. Macrophages are found in lymphoid and in non-lymphoid organs such as liver,
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lung and the peritoneum (Taylor et al.,2005). The peritoneal cavity is a membrane-bound and fluid-
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filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal
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tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T
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cells(Ray &Dittel, 2010). The presence of a high number of unstimulated macrophages in the
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peritoneal cavity makes it a preferred site for the collection of naive tissue resident macrophages.
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Macrophages normally assist in guarding against invading pathogens and regulate tissue remolding.
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Macrophages can identify pathogens by specialized cell membrane receptors (Zhou et al., 2009).
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These receptors enable the macrophages to recognize or differentiate between the viruses, bacteria
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and fungi (MacPherson & Austyn, 2012). Macrophages eliminate these foreign bodies by a process
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known as phagocytosis.
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Phagocytosis is a mechanism of engulfing foreign bodies or large particles in order to support
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the body’s defense (Cannon & Swanson, 1992). Phagocytosis was first observed in starfish larvae by
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Elie Metchnikoff over a century ago. Phagocytosis is present in organisms ranging from unicellular
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microorganisms to specialized cells in higher organisms. In microorganisms, phagocytosis is related
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to food uptake, whereas in multicellular animals, it participates in homeostasis and tissue
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remodeling. Phagocytosis plays an essential role in host-defense mechanisms through the uptake and
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destruction of infectious pathogens and contributes to inflammation and to the immune response
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(García-García & Rosales, 2002).Phagocytosis begins with internalizing the foreign bodyinto
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vesicles known as phagosome. The latter will bind with lysosome to form phagolysosome where the
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digestion of the pathogen occurs via enzymes and toxic peroxides (Jutras & Desjardins,
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2005).Recent studies on different cell systemshave identified the molecules involved in
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phagocytosis.
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Phagocytosis is triggered by the interactionof opsonins, which cover the particle to be
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internalized, with specific receptors on the surface of thephagocyte. These receptors include the Fc
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receptors (FcR), which bind to the Fc portion ofimmunoglobulins (Ig) (Ravetch&Bolland, 2001), and
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the complement receptors (Brown, 1992), which bind to complement onopsonized particles.
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Progressive interaction of the receptors with their ligands allows phagocytosis toproceed in a
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“zipper-like” manner until complete internalization of the particle; which is achieved within
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aspecialized structure, the phagosome. The phagosome then travels inside the cell to fuse
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withlysosomes (Beronet al., 1995) and in this way becomes a microbicidal organelle (Jones et al.,
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1999). Thus, phagocytosis starts with wrapping of the outer macrophage membrane around the
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pathogen and ends up with degrading it (Aderem & Underhill, 1999). Phagocytes are mainly divided
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into professional and non-professional phagocytes. The distinction between the two types of
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phagocytes is based on their ability to carry out this function and if it can digest the foreign body
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(van Oss, 1986). The professional phagocytes have receptors on their surfaces to detect harmful
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objects/foreign bodies which are not normally found in the body(Liarte et al., 2011).
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For this purpose, the senior-level course, Advanced Bioapplications, offered by the
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Department of Biology (DoB) at United Arab Emirates University (UAEU) was designed based on
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experiments that help the students to have more hands on experience and acquire fine skills which
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will support their theoretical studies; and therefore, build their careers (AbuQamaret al., 2015).
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Among these experiments was the in vitro study of phagocytosis by the isolation of mouse cavity
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cells.The phagocytosis process was evaluated in vitro by incubating the isolated and cultured mouse
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cavity cells with antigen particles, such as red blood cells or inactivated yeast cells, for one or two
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hours. Then, the phagocytosis percentage (PP) and phagocytosis index (PI) were determinedunder
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the light microscope to investigate the effectiveness of the mouse immune system. While the
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PPindicates the number of phagocytes present per 100 cells (Campbell et al.,1994), the PI refers to
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the phagocytic activity in which it is measured by counting the number of the engulfed
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microorganism per phagocyte within a period of incubation (O’Brien et al., 2002). In this study, we
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defined a framework to allow active-learning to be incorporated into a large laboratoryBiology
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course in a meaningful and manageable manner.
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Methodology
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Media preparation
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Dulbecco’s modified Eagle's (Harry Eagle) minimal essential medium (DMEM) was prepared in a
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laminar flow hood by adding 10 g of powdered medium (Sigma Aldrich) to 950 mL of sterilized
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distilled water with gentle magnetic stirring. Then, 5.95 g of HEPES buffer (Sigma Aldrich) and 3.7
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g of sodium bicarbonate (Sigma Aldrich) were added. Media additives were 10 mL of
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antibiotic/antimycotic, 10 mL of 50X essential amino acids and 10 ml of 100X non-essential amino
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acids (Gibco-BRL, Life Sciences). The pH of the media was adjusted to 7.4. Finally, the medium
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volume was adjusted to 1000 mL by using sterilized double distilled water.
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The prepared DMEM medium was then sterilized by filtration through 0.22 µm nitrocellulose
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membrane (Nalgene) using a positive pressure air suction. Themedium was kept at 4°C until use.
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Cells were cultured in the prepared DMEM supplemented with 10% Newborn calf serum (Sigma
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Aldrich).
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Isolation of mouse peritoneal macrophages
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The students were divided into four groups; and each group consists of four students. The isolation
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of the mouse peritoneal cavity cells was done as previously described (Ray &Dittel, 2010). Mice
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were sacrified using the anesthesia method followed by cervical dislocation. Mice were obtained
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from the animal facility located in the College of Medicine and Health Sciences, UAEU; and the
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animal study was approved (UAEU-A4/11)by the ethical committee.Four mice were used in the
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experiment. The students followed the research committee guidelines for the care and use of the
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laboratory animals that were kept in stainless steel cages and fed on standard chow and tap water ad
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libitum. Mice were anesthetized by inhaling of diethyl ether in a desiccator that was kept inside
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achemical fume hood. Then, animals were cervically dislocated, transferred to the laminar flow
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hood, sprayed with 70% ethanol and mounted on sterile Styrofoam block on its back. Sterile scissors
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and forceps were used to cut and pull the skin in order to expose the intact peritoneal wall. A 5-mL
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ofcold phosphate buffer saline (PBS) supplemented with 3% newborn calf serum was injected
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carefully into the peritoneal cavity of each mouse using 27 gauge needles in order to harvest the
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cavity cells. After injection, the peritoneum was massaged to allow all the cells to detach from the
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peritoneum cavity into the PBS containing newborn calf serum. By using 5 mL syringes with 25
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gauge needles, the fluids were collected again from the peritoneal cavity of each mouse in collecting
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15-ml sterile tubes that were kept in an ice container. The abdominal cavities of the animals were
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then partially opened to suck as much fluid as possible and added to the cell suspensions in the
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collected tubes. The collected cell suspension was centrifuged at 1,500 rpm for 8 min, then the
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supernatant was discarded and the cell pellet was resuspended in 4mL of DMEM. The cell
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suspension was used for cell counting and viability by the Trypan blue dye (Sigma Aldrich)
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exclusion test before culturing procedures.
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Trypan blue cell viability test
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The 20 µL from each cell suspension was mixed with 20 µL of 0.25% Trypan blue dye (Sigma
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Aldrich) and 10 µL from each cell suspension was applied to the Neubauerhemocytometer (optical
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chamber) by the automatic pipette prior to counting underlight microscope (Leica). The cell count
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was 5 x 105 cellsmL-1. Thus, the total count in the 4 mL cell suspension was 2x 106 cells.
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Culturing procedure
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The isolated mouse peritoneal macrophages were cultured intoNunc Lab-Teck 2 well chamber slides
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at density of 5 x 105 cells/1.5mL of DMEM medium supplemented with 10% Newborn calf serum at
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37°C for 24 hrs in a humidified 5% CO2 incubator.
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Preparation of inactivated yeast
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In order to inactivate the yeast cells and use it as foreign particles for the cultured macrophage,
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Baker's yeast (0.2 g) was added to 15-mL of PBS in a glass test tube. The test tube was autoclaved
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for 15 min at 120oC. After autoclaving, the cells were washed several times withsterile PBS and
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centrifuged at 1,000 rpm for 5 min each time until the phosphate buffer becomes clear. Cleared cells
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were then re-suspended in 10 mLPBS and stored at 4oC(Bos& de Souza,2000).
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Phagocytosis assay
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Adherent peritoneal cells were washed three times with calcium and magnesium free PBS, and
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folded with 1.5 mL of the inactivated yeast and maintained in the CO2 incubator for 2 hrs. After
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incubation, non-phagocytic yeasts were eliminated by washing several times with PBS.
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The adherent peritoneal cellsin the 2-well chamber slides were then fixed with 100%
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methanol (Sigma Aldrich) for 10 min. After fixation, cells were washed with PBS and stained with
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crystal violet for 15 min. The stained cells were washed with distilled water to remove excess stain
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and wereallowed to dry at room temperature. Then, the frames of the stained slides were removed
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and the stained cells were mounted by DPX (Thermo Shandon) and allowed to dry for 24 hrs at
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40°C. Finally, slides were observed by the oil immersion lens of the light microscope to take pictures
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and determine the number of phagocytic cells, non-phagocytic cells and intracellular (engulfed)
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yeasts per macrophage (Bai et al., 2012).
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Statistical analysis
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Statistical analysis was performed for 60 fields in all the prepared cultured-slides whichweretwo
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double chambered slides. Data were expressed as means ± standard deviation (SD) and statistical
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analysis was performed by Student's t-test, where P-value < 0.05 was set as statistical significance.
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EXPERIMENTAL RESULTS
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Isolation of mouse peritoneal cells
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The isolation technique was an important step in this experiment because it allowed the students to
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handle, anaesthetize, treat and dissect laboratory animals and learn how to isolate and maintain
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animal cells. The isolation protocol of mouse peritoneal cells is an essential procedure to study
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phagocytosis. In addition, the peritoneal cavity provides an easily accessible site for harvesting
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resident macrophages (Zhang et al., 2008). This technique is widely used to study macrophage
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biology which can be easily obtained from the peritoneal cavity. In this experiment, the students
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followed the isolation technique previously described (Ray &Dittel, 2010).Cell viability determined
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by trypan blue exclusion staining assay, revealed 100 percent viability of the collected cells hence,
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indicating that the isolation procedure was correctly performed.
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DMEM promotes the peritoneal cells growth
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It has been reported that DMEM and RPMI are the commonly used media for culturing macrophage
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cells isolated from the peritoneal cavity (Bai et al., 2012; Motaet al., 2014; Su et al., 2013; Zhang et
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al., 2008).In this work, DMEM was used for the culture of the isolated mouse peritoneal cells.Phase
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contrast microscopyobservationsrevealed that DMEM promoted the attachment and growth of the
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isolated peritoneal cells. This was clear from the absence of dead suspended cells and the light
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refracted peripheries of the attached cells (Figure 1). In addition, most of the attached cells showed
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pseudopodia-like cytoplasmic extensions and different mitotic stages such as prophase, metaphase,
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anaphase,and telophase (Figure 2).
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Figure 1 Phase contrast observations of isolated mouse peritoneal cells.
Cells were kept in a 37°C incubator with a humidified atmosphere of 5% CO2. Images were obtained
using 20X (panel a) and 40X (panels b-d), showing large sized macrophage nuclei with plenty of
cytoplasm, extensions in cytoplasm and pseudopodia. Panels (c) and (d) show three cell shapes:
extended cells (small arrow), rounded cells (star) and cells with many cytoplasmic extensions (large
arrow).
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Cell morphology examination
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When examined by the inverted microscope, the shape of cells in a culture determines its
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morphology as well astheir health status.Microscopy observation of cultured cells wascrucialforthe
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students sinceitgave them various skills, such as using phase-contrast microscope, identifying
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healthy vs. non-healthy cells, and determining different cell shapes under the microscope. From the
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phase contrast microscopic examinations, students were able to identify that most of the adherent
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cells were macrophage. This was verified by the presence of large sized cell nuclei with plenty of
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cytoplasm, different cytoplasmic extensions and pseudopodia (Figure 1b-d). The pseudopodia are
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usually used for movement; while cytoplasmic extensions are considered markers for phagocytosis.
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Figure 2 Phase contrast images of the proliferating mouse peritoneal cells cultured in DMEM.
Cells were kept at 37°C incubator with a humidified atmosphere of 5% CO2. Images were obtained
using 20X (panel (a)) and 40X (panels (b) to (d)), showing mitotic phases of the in vitro peritoneal as
interphase, prophase, metaphase, anaphase and telophase. Panel (b) shows the formation of cell
clones (circle). Panel (c) shows one cell during cytokinesis (white arrow).
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Furthermore, cell division is consideredas an indicator for the healthy status of the cultured
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cells. In the present work, different stages of mitosis such as metaphase, anaphase and telophase
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(Figure 2b-d)were observed. In addition, cytokinesis leading to two daughter cells was clearly visible
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(Figure 2c).Altogether, these observations clearly attest for the good health of the prepared culture.
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Phagocytosis activity of peritoneal macrophage exposed to inactivated yeast cells
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Students had previously acquired theoretical knowledge about phagocytosis using diagrams or
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animated movies.Here, in order to investigate the phagocytotic process, students isolated mouse
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peritoneal macrophages and incubated them with inactivated yeast at 37°C in an incubator with a
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humidified atmosphere of 5% CO2 for 2hrs (Figure 3).
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Figure3Light micrographs of the mouse peritoneal macrophages.
Cultured peritoneal macrophages were exposed to inactivated yeast cells for 2hrs, fixed by methanol
and stained with crystal violet and examined by oil immersion lens. Panel (a) shows normal
magrophages peritoneal cells spread on a substratum, have large sized nuclei and showing many cell
membrane extensions. Panel (b) shows the peritoneal cells bounded to and phagocytized many yeast
cells. Panel (c) shows a non-phagocytic macrophage. Panels (d, e and f) illustrate all the
phagocytosis stages; binding, phagophore and phagosome and finally phagolysosme, respectively.
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Students were able to detect phagocytosis at different stages. Indeed, early steps of
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phagocytosis were clearly identified by the detection of yeast cells bound to macrophages (Figure
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3c). Later stages of phagocytosis were identified by the presence of cytoplasmic membrane-
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surrounded vesicles (phagosomes) containing engulfed yeast (Figure 3d). Phagolysosomes, the site
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of enzymatic degradation of the yeast cell,resulting from the fusion of the phagosomes and
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lysosomes also were clearly visible (Figure 3e). Phagocytosis was also measured, by calculating the
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phagocytosis percentage (PP), as previously described (Methodology).The percentage of phagocytic
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and non-phagocytic cells was 80.55% ± 0.16 and 20.44% ± 0.16, respectively(Figure 4).
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Figure 4 The phagocytosis percentage of phagocytic and non-phagocytic cells of cultured cells.
The phagocytosis percentage was calculated in 60 fields by counting the phagocytic cell in each field
and then it was divided by the total number of cells present in the same field. There was significant
change between phagocytic and non- phagocytic cells (p<0.05).
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In order to investigate whether the phagocytosis process was efficient in our system, we
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determined another phagocytosis parameter which is the phagocytosis index (PI). The PI was
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calculated by dividing the number of yeast cells that were bound and engulfed by the peritoneal
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macrophages over the total number of phagocytizing macrophages. We found that the PI was 4.29
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yeast cells per macrophage (Table 1) within 2 hours. Altogether, our data suggest that
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micepossessesan efficient immune system that can eliminate foreign particles in a short time
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consideringthe total numberof phagocytic cells which include neutrophils, eosinophils, basophils,
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monocytes, macrophages and dendritic cells in the animal body.
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DISCUSSION
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A phagocytosis experiment was performed by undergraduate senior students during the capstone
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course “Advanced Bioapplications”in the Department of Biologyat UAEU. The experiments were
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conducted in three sessions during one week of this practical course. The phagocytosis experiment
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taught the students a number of skills that are related to their
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Table 1. The phagocytosis index (PI) which refers to the number of yeast cells that were
engulfed by the macrophages within 2-hrs in all the examined fields (mean: 4.29±1.64).
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Field #
PI
Field #
PI
Field #
PI
Field #
PI
1
3.59
16
3.11
31
4.13
46
5.29
2
2.73
17
5.10
32
5.37
47
6.00
3
2.57
18
3.50
33
3.91
48
8.43
4
2.82
19
4.66
34
2.55
49
7.63
5
2.46
20
2.75
35
2.10
50
5.23
6
2.38
21
3.20
36
4.58
51
5.83
7
2.63
22
8.00
37
2.87
52
5.71
8
2.56
23
4.90
38
3.70
53
5.00
9
3.63
24
2.50
39
4.50
54
4.60
10
2.00
25
4.55
40
3.62
55
4.75
11
3.78
26
6.89
41
4.92
56
9.75
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2.57
27
4.11
42
3.40
57
5.70
13
3.50
28
2.54
43
4.14
58
4.40
14
2.70
29
5.07
44
3.67
59
6.73
15
3.50
30
5.38
45
4.91
60
4.80
educationalknowledge.Scientifically, this experiment increasedthe students understanding about
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phagocytosis process, including the phagocytic cells, stages of phagocytosis, and phagocytosis
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parameters. Moreover, they learned different experimental techniques such as experimentaldesign,
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mediapreparation, animal handling and dissection, statistical analysis and datainterpretation. The
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experiment also investigated the efficiency of the mouse immune system and correlated their
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theoretical knowledge with real experiment conducted by them.
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The immune system carries out various defense processes in order to protect the animal form
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as
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macrophages(Medzhitov, 2008). These macrophages have an essential role in the body defense
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especially in phagocytosis and are considered as professional phagocytic cells. Macrophages first
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recognize the foreign body and then start to engulf it. Herein, we investigated the efficiency of the
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mouse immune system by measuring the phagocytosis percentage (PP) and phagocytosis index (PI)
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of its peritoneal macrophages in vitro by using inactivated yeast cells as foreign particulates (Gagnon
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et al., 2002).It is well known that high percentage of phagocytic cells is an indication of an efficient
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immune system (Campbell et al.,1994). Our results showed that the PP was about 81% indicating
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that most of the cultured macrophages were phagocytosis-competent (Figure 4). Moreover, the
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phagocytosis index showed that approximately 4 yeast cells were engulfed by each macrophage cell
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in 2 hrs. Together, our datasuggest a high efficiency of the mouse immune system.
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foreign
bodies.
This
isdone
by
recruiting
many
different
types
of
cells
such
Similar approach to the one used in this study was carried by another group (Bos& de
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Souza,2000), where they describeda method by which internalized and surface-bound yeast particles
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can be identified by differential interference contrast microscopy using Trypan blue to stain surface-
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bound yeast particlesin order to examine phagocytosis process and the receptors involved. In that
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report, they also used mouse peritoneal macrophages with yeast cells and found that the percentage
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of internalized yeast was 69%, which is lower to the present work (80.5%). Moreover, the
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internalized yeast particle was 4.1 yeast cell/ macrophage which is in agreement with our finding (4
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yeast/macrophage).Ourresultsalso revealed thatDMEM is the most appropriate medium because
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cultured peritoneal cells were able toattach quickly and proliferate as well. This finding is in
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agreement with previous work by Anwar and collaborators who also cultured in DMEM,harvested
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and plated the peritoneal cavity macrophages and J774 cell line macrophage (Anwar et al., 2009).
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Similarly, Motaet al.,(2014)used DMEM as culture media for mouse peritoneal macrophages.
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Flow cytometry and fluorescence microscopy were usedto determine the PP and PI for
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different Candida species exposed in vitro to isolate mouse peritoneal macrophages(Carneiro et al.,
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2014). The percentage of macrophages that engulfed or attached to C. orthopsilosis was almost 80%.
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The result matches and supports our datadespitethe slight variationbetween the two pathogenic
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species. It is noteworthy to mention that several factors might influence the phagocytosis processes
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such as the size ofpathogens and the type of the cells involved mouse macrophages and neutrophils
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against C. albicanswere comparedusing adherent monolayer method(Vonket al., 2002). They found
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that macrophages had higher PP where it reached approximately 55% compared with neutrophils
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which reached 40% only. Another study showed that human neurophils had different PPs when
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exposed to two different species of yeast which are C. parapsilosis and C. albicans(Destin et al.,
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2009). The PPs were only 20% when the neutrophils exposed to C. albicans, whereas it reached 80%
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when treated with C. parapsilosis.
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CONCLUSIONS
In conclusion, this study revealedthat the mouse immune system is a highly efficient system
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that can eliminate pathogens through phagocytosis. We found that such types of experiments
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increase the hands on skills,and support and encourage the students to participate in research and
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create a friendly-learning environment laboratorycourses. As potential futurescientists and/ordecision
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makers, students are required to be able to conduct and plan researches and write scientificreports.
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ACKNOWLEDGEMENT
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We would like to express our thanks and appreciations to all students from Advance Bioapplications
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course for their participation in conducting the phagocytosis experiment.This project was funded by
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the National Research Foundation-UAEU [RSA 2014-06]; and the Khalifa Center for Biotechnology
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and Genetic Engineering-UAEU [KCGEB-2-2013] to SAQ.
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REFERENCES
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Aderem, A.,& Underhill, D.M. (1999). Mechanisms of phagocytosis in macrophages. Annual Review
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Anwar, D., Keating, A., Joung, D., Sather, S., Kim, G., Sawczyn, K., Brandao, L., Henson, B.,&
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