Actin Expression in Hela Cells by APAAP Immunocytochemistry

Actin Expression in Hela Cells by APAAP Immunocytochemistry Kunthavai Jeevananthan Isolation of mononuclear cells, cell counts, cell staining and immunochemistry Aims: to test the morphology of sheep’s blood smear and to calculate the number of viable cells and the percentage viability of live and dead mononuclear cells. To investigate and analyse actin expression in Hela cells by APAAP immunocytochemistry. There are three main components of blood these include erythrocytes (red blood cells), leukocytes (white blood cells) and thrombocytes (platelets). Leukocytes are produced and store in a number of locations called lymphoid organs inside the body such as the spleen, thymus and the bone marrow through a process known as haematopoiesis. They are part of the bodies primary defence mechanism against infections caused by disease causing microorganisms entering the body. Leukocytes are constantly produced and released into the bloodstream where they circulate the body travelling from organs to lymph nodes where they are stored via the lymph and blood vessels. There are three different types of leukocytes known as granulocytes, lymphocytes and monocytes (Kara, 2011). The granulocytes include eosinophils and basophils which are involved in immune responses and also neutrophils ingest and kill any foreign body that enter the body such as bacteria and fungi. Lymphocytes help to protect against viral infections whereas others develop into antibodies against foreign invasions. Blood smears and differential staining techniques such as the Giemsa and Grunwald staining can be conducted to analyse the morphology of blood cells to help diagnose blood disorders. Mononuclear cells also known as agranulocytes are white blood cells containing one lobed nucleus. These include monocytes and immunocytes such as B-lymphocytes, T-lymphocytes, natural killer (NK) cells and macrophages. Normal blood values vary from around 50, 00 to 10, 000 leukocytes per cubic millimetre. High values of leukocytes are known as leucocytosis and it is most commonly due to inflammations as a result of an immune response or blood diseases such as leukaemia (Marshall, 2008). Low values of leukocytes are known as leukopenia this occurs when the bone marrow is unable to produce sufficient RBC like for example neutrophils which leads to neutropenia, this can make the body more prone to diseases and infections therefore mononuclear cell counts are widely used in immunology, haematological malignancy, oncology and vaccine development to monitor immunological functions and to identify and diagnose blood related conditions. This can be achieved by the Ficoll-Hypaque technique; a density gradient method that separates lymphocytes from other formed elements in blood which can be removed and stained with trypan blue in order to be viewed on a haemocytometer under the microscope. Live cells have an intact cell membrane that is impermeable to certain dyes such as trypan whereas dead cells do not therefore viable cells with a clear cytoplasm and nonviable cells with a blue cytoplasm can be identified and calculated (Harlad and Heinz et al, 2004). Immunocytochemistry is the identification of a tissue by specific antigen-antibody interaction where the antibodies are marked with a visible label for examination under the microscope which was first described by Albert H. Coons in 1941. This technique was further developed in different ways by using different indicator molecules such as fluorescein molecules, biotin and enzymes i.e. peroxidase. APAAP (alkaline phosphatase anti-alkaline phosphatase) is an alkaline phosphatase method first described by Cordell et al in 1984 (John, 2008). Antibodies are proteins known as immunoglobulins (Igs) which are further categorised in to different isotypes. The IgG isotype is most commonly used in immunocytochemistry as its biding is more consistent. Actin is a highly conserved and abundant cytoskeletal protein which exists in six different known isofroms in mammals therefore it can be used to detect cancer cells like Hela cells, a continuously cultured strain isolated from human cervical cance r cells, as they are rapidly multiplying and are rich in Actin. Beta and gamma actin are known as cytoplasmic actin as they are expressed in non-muscle cells. Mouse anti-actin antibody is used in this technique as they are a form of highly conserved globular proteins which are found in cell mortality, structure and integrity of all eukaryotic cells hence are often found around the nucleus in the cytoplasm (Richard, 2010). Results Figure 1 Figure 1.1 Red blood cell Platelets Figure 2Figure 2.1 Figure 3: Hela cells only Figure 4: No Primary control Figure 5: Isotype control Figure 6: Actin Discussion The Giemsa and Grunwald staining of the sheep’s blood smear show the all the red blood cells are approximately the same shape and size however the number of red blood cells present appears to be fairly lower than expected. There are a number of reasons as to why this could have occurred such as lack of iron lead to failure of haemoglobin production therefore the number of red blood cells lost cannot be replaced efficiently. Other causes that may result in decreased numbers of erythrocytes may be due to bone marrow damage or disorders, internal or external bleeding and kidney failure. The red blood cells were all however the same shape and size which shows that there is no cause of concern for diseases related to abnormal shapes of RBCs such as sickle cell anaemia. Although the cells appear spherical in figure 1 this may be due to the low magnification and resolution therefore in order to confirm spherocytosis further analysis in higher resolution and tests need to be carried o ut. The light pink staining in the centre of the RBC shows that the area is less dense indicating the absence of a nucleus and the biconcave structure. The mononuclear cell count shown in figure 2 does not show any live mononuclear cells. There are a number of reasons as to why this may have occurred like for example when pipetting the separated white blood cells layer on top of the ficoll layer may have been taken from the wrong layer of cells. Another point to taken into consideration is that when removing the supernatant the live cells may have also been accidentally removed with it. As sheep’s blood cells were used instead of human blood the layering in ficoll is likely to be slightly different therefore in order to obtain a better sample of live and dead mononuclear cells the white blood cells should be removed closer to the bottom of the tube just above the red blood cell pallet at the bottom. In figure 2 there were no dead cells present in the centre square therefore the percentage viability cannot be calculated by dividing the number of live cells by the number of dead cells. The red blood cell count varies from perso n to person as factors such as the altitude and gender. The normal value of male blood count is 4.5 to 6.0 x106 and in females it is 4.0 and 4.5 x 106 therefore when comparing the sheep’s RBC count of 9.3 x 106 calculated from figure 2 is high. A high number of red cell counts may be due to lung diseases, kidney tumours and polycythaemia vera (PVC). The APAAP immunocytochemistry method conducted shows that the Hela cells are a single lineage as all cells in figure 6 were stained brown when incubated with primary anti-actin antibody and secondary polyclonal Goat anti-mouse antibody which are complimentary to each other hence they will only bind to the same cell lineage with the specific antigen. Figure 5 shows a positive isotype control of hela cells incubated with purified mouse monoclonal. This confirms that the actin antibody specific background staining shown in figure 5 were not due to non-specific interactions of antibody molecules with the HeLa cell sample. Figure 4 also shows that hela cells without the secondary antibody alone without the primary antibody convey no significant background staining caused by DAB hence also supporting the fact the background staining is highly antigen specific (Stross and Jones et al, 1989). This method can be used to analyse the cellular contents, localisation and the distribution of cells in their surroundings and their relationship with other cells in peripheral blood smears, swabs, cell cultures, histological biopsy and bone marrow aspirates hereby giving definitive diagnosis to tumour and cancer diagnosis. An alternative immunocytochemistry method is Avidin-Biotin complex (ABC). Similarly like the APAAP method it involves the application of a primary antibody, secondary antibody however APAAP complex is replaced with Avidin-Biotin peroxidase complex (Buchwalow and Bocker, 2010). Reference Buchwalow, I. B, Bocker. W. (2010). Immunocytochemistry: Basics and method: Immunostaning enchantment. 48-52. Germany. Springer-Verlag. Harald, T., Heinz, D., Trosten, H. (2004). Colour Atlas of haematology: practical microscopic and clinical diagnosis. 63-66. German. Georg Thieme Verlag. John, B. D. (2008). Theory and practice of histological techniques: 433-438. United States of America. Elsevier Limited. Kara, R. (2011). Blood: Physiology and circulation. 16-22. New York. Britannia educational publishing. Marshall, C. C. (2008). Diseases and Disorders: Blood diseases. 103-106. Malaysia. Library congress Catalkukuoging-in-publication-data. Richard, B. W. (2010). Immunocytochemistry: A practical guide to biomedical research. 1-10. United States of America. Springer science and business media. Stross, P. W., Jones, M., Manson, D. Y. (1989). Automation of APAAP immunocytochemical techniques J. clin Pathol. 42(1), 106-112.

The Whiskey Rebellion Essay -- History USA Historical Essays

The Whiskey Rebellion CONTENTS 1. Introduction to the French and Indian War 2. Domestic and social differences in the region 3. Washington?s statement 4. Attack on the Lys 5. Battle for the Fort Lydius 6. Battle for Forts William Henry and Bull 7. Battle for Fort Oswego 8. Battle for Quebec 9. Treaties Senecas and Paris   Ã‚  Ã‚  Ã‚  Ã‚  The Whiskey Rebellion of 1794 helped bring about the demise of the aristocratic Federalist Government in favor of the democratic Republican Government, concerned with the needs of all of its citizens. The new country of the United States of America suffered many growing pains in trying to balance its commitment to liberty with the need for order. How much control is enough and what will be too much? After the Revolutionary War, the country purposely did not have a strong central government (that's what we fought against with the British). The states did as they pleased because the Articles of Confederation in 1781 gave them every power, jurisdiction and right not expressly delegated to the Continental Congress. Congress had no power to tax, regulate commerce, draft troops, or enforce foreign treaties. It was mainly a friendly overseer: thus the expression "the Do-Nothing Congress." Each state considered itself sovereign, free and independent, and easterners and westerners were separated by geography as well as their own concerns.   Ã‚  Ã‚  Ã‚  Ã‚  To make matters worse, Spain and Britain were wreaking havoc along our borders. British troops, violating the Treaty of Paris, refused to vacate their garrisons along the Great Lakes; Spain, who held New Orleans, closed the Mississippi River to American shipping below Nachez and actively encouraged American settlers to break away from the Union and establish relations with them; Westerners in Kentucky, Tennessee, Virginia and Pennsylvania were subjected to attacks by marauding Indians (often instigated by the Spanish and British). Congress did not have the power to send troops for defense or protection, and the easterners in these states were too busy with politics to worry about their western frontiersmen. Consequently, the westerners did as they pleased with no regard to the laws the easterners made.   Ã‚  Ã‚  Ã‚  Ã‚  States had the power to levy taxes. Massachusetts imposed hefty taxes to help pay off its war debts. With the postwar depression, many farmers had trouble paying their mortgages... ...ependent people west of the mountains to submit to Federalist principles, as the unwillingness of the farmers of the Shays Rebellion, helped publicize some of the problems the settlers were having and allowed citizens to voice their disapproval of their government without being considered treasonous. Two of the rebels in the Whiskey Rebellion were tried for treason, convicted, and then pardoned by President Washington. Thomas Jefferson condemned the use of military force and Hamilton's misuse of federal power. Hamilton's vision of an aristocracy for the federal government was fortunately put to rest as the Republican-Democrat Party replaced it and evolved into the government that we have today.   Ã‚  Ã‚  Ã‚  Ã‚   Works Cited 1.  Ã‚  Ã‚  Ã‚  Ã‚  Brinkley, Alan. "The Unfinished Nation." McGraw-Hill, New York. 2000. 150-170. 2.  Ã‚  Ã‚  Ã‚  Ã‚  Kauffman, Bruce. "Viewpoint The Whiskey Rebellion: Taxing "Sin" Then and Now." The Early American Review. Fall 1996. http://www.earlyamerica.com/review/fall96/whiskey.html. 3.  Ã‚  Ã‚  Ã‚  Ã‚  Maddox, Robert James. "American History Volume I." Dushkin/McGraw-Hill, New York. 1999. 72-81. 4.  Ã‚  Ã‚  Ã‚  Ã‚  Specht, Elmer E. "The Whiskey Rebellion of 1974. http://capo.org/opeds/whiskey.html.

International Journal Of Engineering Research and General Science ISSN-2091-2730

International Journal of Engineering Research and general science (ISSN 2091-2730), Impact factor (3.72 ICV) is an open access peer review publication which is established for publishing the latest trends in engineering. We give priority to quality papers which emphasis on basic and important concept through which there would be remarkable contribution to the research arena and also publish the genuine research work in the field of science, engineering and technologies.This publication welcomes all the original research papers, conceptual articles, reviewed papers from tremendous fields of science and technology and moreover papers are selected and are reviewed by our expert editorial team as per the paper forwarded by assistant editorial in chief. Our strong team member will come back to you with all the feedback within a week.The papers so accepted are to undergo certain criteria of P&R publication and finally it will be in online with in a week. Any research which are carried on f or the prompt impact to the society and which also contribute for environmental conservation are given special priority.Call For Research Papers – Vol.2 Issue 2It's our privilege to invite you to submit your research work in our journal which is influencing the world with the concept of innovation and techniques. You can send your paper on any of this discipline1. Civil engineering, Mechanical Engineering, Computer science Engineering, Electrical Engineering, Information Technology, Electronic and communication engineering, aeronautical engineering, General engineering, resources engineering and other tremendous discipline of engineering field.2. Medicine, Microbiology, Biotechnology, Pharmacy, general biology, Physic, Chemistry, Mathematics and other field of general science.You can send your Manuscripts to our asso editor in chief via email- [email  protected], [email  protected]PROCESS Of REVIEWOur review process is double-blinded that is the reviewer does not know the author(s) detail and meanwhile author(s) also are unaware about the reviewer. Authors are finally provided with the feedback of the paper.AIM & SCOPEThis journal is commenced with the aim of assisting the researchers to provide the best platform to expose their innovation and thoughts. we appreciate the new vision and innovative thoughts that helps in research and findings.We also aims to commence own research platform through which tremendous innovation will be carried on very soon.For Detail- www.ijergs.org

Financial Innovation And Its Effect On The Financial Crisis

Another problem connected to the securitization of loans is the fact that the required off-balance sheet transactions increase the difficulty of determining a company’s true leverage. While off-balance sheet transactions are not necessarily a bad thing, they do disguise the true debt of a company in that they make it appear as if the obligation to repay the security investors falls on the SPV rather than the originator (Pala). Furthermore, a movement of assets to an SPV can result in the appearance of a reduction of leverage, which may make a company appear more attractive to investors, or in the case of a bank, allow them to decrease their capital requirements when they have not in fact de-levered and have simply moved items off of their†¦show more content†¦Thus, the novelty and complexity of these new securities can result in grave misunderstandings about not only the functionality, but also the riskiness of certain forms of securitization. Involvement in the 2008 Financial Crisis The 2008 financial crisis is complicated, as are its many causes, however it is clear that poor understanding and lack of regulation of securitization played a significant role in bringing about the crisis. The problems began when housing prices started to decline in 2006, resulting in an unexpected increase in mortgage defaults as homeowners found themselves owing more on their mortgage than their houses were worth. When mortgages began to default, the collateral on mortgage-backed securities lost its value, which resulted in the failure of banks, GSEs and funds that had invested heavily in mortgage backed securities, and another related offerings (Schulz). 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