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Cell Cycle Analysis
Cell cycle analysis by DNA content measurement is a method that most frequently employs flow cytometry to distinguish cells in different phases of the cell cycle. Before analysis, the cells are usually permeabilised and treated with a fluorescent dye that stains DNA quantitatively, such as propidium iodide (PI) or 4,6-diamidino-2-phenylindole (DAPI). The fluorescence intensity of the stained cells correlates with the amount of DNA they contain. As the DNA content doubles during the S phase, the DNA content (and thereby intensity of fluorescence) of cells in the G0 phase and G1 phase (before S), in the S phase, and in the G2 phase and M phase (after S) identifies the cell cycle phase position in the major phases (G0/G1 versus S versus G2/M phase) of the cell cycle. The cellular DNA content of individual cells is often plotted as their frequency histogram to provide information about relative frequency (percentage) of cells in the major phases of the cell cycle. Cell cycle a ...
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Flow Cytometry
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so light is absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer. Flow cytometry is routinely used in basic research, clinical practice, and clinical trials. Uses for flow cytometry include: * Cell counting * Cell sorting * Determining cell characteristics and function * Detecting microorganisms * Biomarker detection * Protein engineering detection * Diagnosis of health disorders such as blood cancers * Measuring ...
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University Of California
The University of California (UC) is a public land-grant research university system in the U.S. state of California. The system is composed of the campuses at Berkeley, Davis, Irvine, Los Angeles, Merced, Riverside, San Diego, San Francisco, Santa Barbara, and Santa Cruz, along with numerous research centers and academic abroad centers. The system is the state's land-grant university. Major publications generally rank most UC campuses as being among the best universities in the world. Six of the campuses, Berkeley, Davis, Irvine, Los Angeles, Santa Barbara, and San Diego are considered Public Ivies, making California the state with the most universities in the nation to hold the title. UC campuses have large numbers of distinguished faculty in almost every academic discipline, with UC faculty and researchers having won 71 Nobel Prizes as of 2021. The University of California currently has 10 campuses, a combined student body of 285,862 students, 24,400 faculty members, 1 ...
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5-ethynyl-2'-deoxyuridine
5-Ethynyl-2′-deoxyuridine (EdU) is a thymidine analogue which is incorporated into the DNA of dividing cells. EdU is used to assay DNA synthesis in cell culture and detect cells in embryonic, neonatal and adult animals which have undergone DNA synthesis. Whilst at high doses it can be cytotoxic, this molecule is now widely used to track proliferating cells in multiple biological systems. EdU-labelling allows cells to be isolated without denaturing RNA, allowing researchers to determine the transcriptional profile of cells. This approach has been used to assess transcription in neuronal cells and tissues that have recently divided either ''in vitro'' or ''in vivo''. Detection EdU is detected with a fluorescent azide which forms a covalent bond using click chemistry. Unlike the commonly used bromodeoxyuridine, EdU detection requires no heat or acid treatment. EdU incorporated into DNA induces DNA damage, primarily during DNA replication, which is reflected by phosphorylation of ...
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5-bromo-2'-deoxyuridine
Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU, BUdR, BrdUrd, broxuridine) is a synthetic nucleoside analogue with a chemical structure similar to thymidine. BrdU is commonly used to study cell proliferation in living tissues and has been studied as a radiosensitizer and diagnostic tool in people with cancer. During S phase of the cell cycle (when DNA replication occurs), BrdU can be incorporated in place of thymidine in newly synthesized DNA molecules of dividing cells. Cells that have recently performed DNA replication or DNA repair can be detected with antibodies specific for BrdU using techniques such as immunohistochemistry or immunofluorescence. BrdU-labelled cells in humans can be detected up to two years after BrdU infusion. Because BrdU can replace thymidine during DNA replication, it can cause mutations, and its use is therefore potentially a health hazard. However, because it is neither radioactive nor myelotoxic at labeling concentrations, it is widely preferred f ...
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Cyclin B1
G2/mitotic-specific cyclin-B1 is a protein that in humans is encoded by the ''CCNB1'' gene. Function Cyclin B1 is a regulatory protein involved in mitosis. The gene product complexes with p34 (Cdk1) to form the maturation-promoting factor (MPF). Two alternative transcripts have been found, a constitutively expressed transcript and a cell cycle-regulated transcript that is expressed predominantly during G2/M phase of the cell cycle. The different transcripts result from the use of alternate transcription initiation sites. Cyclin B1 contributes to the switch-like all or none behavior of the cell in deciding to commit to mitosis. Its activation is well-regulated, and positive feedback loops ensure that once the cyclin B1-Cdk1 complex is activated, it is not deactivated. Cyclin B1-Cdk1 is involved in the early events of mitosis, such as chromosome condensation, nuclear envelope breakdown, and spindle pole assembly. Once activated, cyclin B1-Cdk1 promotes several of the events of ea ...
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Cyclin A
Cyclin A is a member of the cyclin family, a group of proteins that function in regulating progression through the cell cycle. The stages that a cell passes through that culminate in its division and replication are collectively known as the cell cycle Since the successful division and replication of a cell is essential for its survival, the cell cycle is tightly regulated by several components to ensure the efficient and error-free progression through the cell cycle. One such regulatory component is cyclin A which plays a role in the regulation of two different cell cycle stages. Types Cyclin A was first identified in 1983 in sea urchin embryos. Since its initial discovery, homologues of cyclin A have been identified in numerous eukaryotes including ''Drosophila'', ''Xenopus'', mice, and in humans but has not been found in lower eukaryotes like yeast. The protein exists in both an embryonic form and somatic form. A single cyclin A gene has been identified in Drosophila while Xe ...
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Cyclin E
Cyclin E is a member of the cyclin family. Cyclin E binds to G1 phase Cdk2, which is required for the transition from G1 to S phase of the cell cycle that determines initiation of DNA duplication. The Cyclin E/CDK2 complex phosphorylates p27Kip1 (an inhibitor of Cyclin D), tagging it for degradation, thus promoting expression of Cyclin A, allowing progression to S phase. Functions of Cyclin E Like all cyclin family members, cyclin E forms a complex with cyclin-dependent kinase (CDK2). Cyclin E/CDK2 regulates multiple cellular processes by phosphorylating numerous downstream proteins. Cyclin E/CDK2 plays a critical role in the G1 phase and in the G1-S phase transition. Cyclin E/CDK2 phosphorylates retinoblastoma protein (Rb) to promote G1 progression. Hyper-phosphorylated Rb will no longer interact with E2F transcriptional factor, thus release it to promote expression of genes that drive cells to S phase through G1 phase. Cyclin E/CDK2 also phosphorylates p27 and p21 during ...
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Cyclin D1
Cyclin D1 is a protein that in humans is encoded by the ''CCND1'' gene. Gene expression The CCND1 gene encodes the cyclin D1 protein. The human CCND1 gene is located on the long arm of chromosome 11 (band 11q13). It is 13,388 base pairs long, and translates into 295 amino acids. Cyclin D1 is expressed in all adult human tissues with the exception of cells derived from bone marrow stem cell lines (both lymphoid and myeloid). Protein structure Cyclin D1 is composed of the following protein domains and motifs: * retinoblastoma protein (pRb) binding motif; * cyclin box domain for cyclin-dependent kinase (CDK) binding and CDK inhibitor binding; * LxxLL binding motif for co-activator recruitment; * PEST sequence that may mark the protein for degradation; * threonine residue (threonine 286) that controls nuclear export and protein stability. Function The protein encoded by this gene belongs to the highly conserved cyclin family, whose members are characterized by a dramatic ...
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Acridine Orange
Acridine orange is an organic compound that serves as a nucleic acid-selective fluorescent dye with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows the dye to interact with DNA by intercalation, or RNA via electrostatic attractions. When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein. Acridine orange and fluorescein have a maximum excitation at 502 nm and 525 nm (green). When acridine orange associates with RNA, the fluorescent dye experiences a maximum excitation shift from 525 nm (green) to 460 nm (blue). The shift in maximum excitation also produces a maximum emission of 650 nm (red). Acridine orange is able to withstand low pH environments, allowing the fluorescent dye to penetrate acidic organelles such as lysosomes and phagolysosomes that are membrane-bound organelles essential for acid hydrolysis or for producing products of phagocytosis of apopt ...
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The Journal Of Cell Biology
The ''Journal of Cell Biology'' is a peer-reviewed scientific journal published by Rockefeller University Press. History In the early 1950s, a small group of biologists began to explore intracellular anatomy using the emerging technology of electron microscopy. Many of these researchers were at The Rockefeller Institute of Medicine, the predecessor of The Rockefeller University. As their work progressed to publication, they were disappointed with the limited quality of halftone image reproduction in the printed journals of the time, and frustrated by the narrow editorial policies of existing journals regarding their image-based results. In 1954, the Director of the Rockefeller Institute, Detlev Bronk, convened a luncheon to discuss the creation of a new journal as a venue for publication of this type of work. The first issue of ''The Journal of Biophysical and Biochemical Cytology'' was published less than a year later on January 25, 1955. A subscription cost $15 per year. The l ...
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Harvard Medical School
Harvard Medical School (HMS) is the graduate medical school of Harvard University and is located in the Longwood Medical Area of Boston, Massachusetts. Founded in 1782, HMS is one of the oldest medical schools in the United States and is consistently ranked first for research among medical schools by '' U.S. News & World Report''. Unlike most other leading medical schools, HMS does not operate in conjunction with a single hospital but is directly affiliated with several teaching hospitals in the Boston area. Affiliated teaching hospitals and research institutes include Dana–Farber Cancer Institute, Massachusetts General Hospital, Brigham and Women's Hospital, Beth Israel Deaconess Medical Center, Boston Children's Hospital, McLean Hospital, Cambridge Health Alliance, The Baker Center for Children and Families, and Spaulding Rehabilitation Hospital. History Harvard Medical School was founded on September 19, 1782, after President Joseph Willard presented a report with ...
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