Cell Cycle Analysis & Proliferation Methods PDF

Cell Biology Methods for assessing Cell Events: Cell cycle analysis, cell Proliferation, and apoptosis Presented by Dr Ghada KHAWAJA Cell cycle analysis, cell Proliferation and apoptosis The balance of cell proliferation an...

Cell Biology Methods for assessing Cell Events: Cell cycle analysis, cell Proliferation, and apoptosis Presented by Dr Ghada KHAWAJA Cell cycle analysis, cell Proliferation and apoptosis The balance of cell proliferation and apoptosis is important for both development and normal tissue homeostasis. Cell proliferation is an increase in the number of cells as a result of growth and division. Cell proliferation is regulated by the cell cycle, which is divided into a series of phases. Apoptosis, or programmed cell death, results in controlled self-destruction. Several methods have been developed to assess apoptosis, cell cycle, and cell proliferation for applications from basic research to drug screening. Over the years, multicolor flow cytometry has become essential in the study of apoptosis, cell cycle, and cell proliferation. Success of the technology results from its ability to monitor these processes along with other cellular events, such as protein phosphorylation or cytokine secretion, within heterogeneous cell populations.  The Cell Cycle The cell cycle has two major phases: interphase, the phase between mitotic events, and the mitotic phase, where the mother cell divides into two genetically identical daughter cells. Interphase has three distinct, successive stages. During the first stage called G1, cells “monitor” their environment, and when the requisite signals are received, the cells synthesize RNA and proteins to induce growth. When conditions are right, cells enter the S stage of the cell cycle and “commit” to DNA synthesis and replicate their chromosomal DNA. Finally in the G2 phase cells continue to grow and prepare for mitosis. Cell Cycle Analysis using Flow Cytometry The most common method for assessing the cell cycle is to use flow cytometry to measure cellular DNA content. Using this method, different treatments can be assessed for their effect on cell cycle for example, through analyzing the proportions of cells in different stages of the cell Cycle. Before analysis, the cells are permeabilised and treated with a fluorescent dye that stains DNA quantitatively, usually propidium iodide (PI). Using a flow cytometer, labeled cells suspended in a liquid stream are passed through a laser light beam in a single file fashion so that the fluorescence intensity emitted of each cell is measured. The fluorescence intensity of stained cells at certain wavelengths will therefore correlate with the amount of DNA they contain. As the DNA content of cells duplicates during the S phase of the cell cycle, the relative amount of cells in the G0 phase and G1 phase (before S phase), in the S phase, and in the G2 phase and M phase (after S phase) can be determined, as the fluorescence of cells in the G2/M phase will be twice as high as that of cells in the G0/G1 phase.  Cell Cycle Analysis: Flow Cytometry Experimental procedure: The first step in preparing cells for cell-cycle analysis is permeabilization of the cells' plasma membranes. This is usually done by incubating them in a buffer solution containing a detergent such as Triton X-100 , or by fixating them in ethanol. Most fluorescent DNA dyes are not membrane permeable, that is, unable to pass through an intact cell membrane. Permeabilization is therefore crucial for the success of the next step, the staining of the cells. Prior to or during the staining step, the cells will usually be treated with RNase A to remove RNAs from the cells. This is important because dyes that stain DNA will also stain RNA, thus creating artefacts that would distort the results. Aside from propidium iodide, quantifiable dyes that are frequently used include (but are not limited to) DRAQ5, 7-Aminoactinomycin D, DAPI and Hoechst 33342. When the cells pass through the flow cytometer's laser, a fluorescence pulse is generated that correlates with the amount of dye associated with the DNA and thus with the total amount of DNA in the cell. The distribution of the cells into their respective cell-cycle phases is based on their DNA content: sub-G0/G1 cells were less than 2n, G0/G1 cells were 2n, S/M phase cells were >2n. The cell death is determined by an increase in cells in the pre-G phase as compared to the control.  Tools and Techniques to Study Cell Proliferation Cell proliferation can occur in response to many stimuli such as cytokine exposure or a variety of other processes. Tools for VPD450 Analysis Violet Proliferation Dye 450 can be used for the detection of cell proliferation with the violet laser using multicolor flow cytometry. VPD450 is a nonfluorescent esterified dye. The ester group allows the dye to enter the cell. Once the dye is inside the cell, esterases cleave off the ester group to convert the dye into a fluorescent product and trap it inside the cell. With each replication event the amount of dye in the cell is decreased, leading to a characteristic pattern. Tools for BrdU Analysis A series of antibodies and kits designed for the detection of proliferating cells by measurement of bromodeoxyuridine (BrdU), an analog of the DNA precursor thymidine used to measure de novo DNA synthesis. During the S phase of the cell cycle (DNA synthesis) BrdU is incorporated into the newly synthesized DNA and can be readily detected by anti-BrdU specific antibodies. The antibodies and kits designed for the detection of BrdU are available for both intracellular flow cytometry and immunohistochemistry. Tools and Techniques to Study Cell Proliferation In addition to DNA increases, levels of certain proteins also rise as a result of cell proliferation. For example, Ki67 is an antigen that is expressed in the nucleus of dividing cells.However, during the G0 phase of the cell cycle it is not detected. Ki67 can be combined with other proliferation markers such as BrdU and VPD450 for added confidence. These markers can also be combined with cell surface and other types of markers to gain additional information about cell subsets and their signaling pathways. The MTT-based assay can be also used to measure cell proliferation. This assay is based on the ability of the mitochondria of metabolically active cells to cleave the yellow tetrazolium salt MTT to purple formazan crystals. The formazan crystals are then rendered soluble using a solubilizing solution and measured spectrophotometrically using ELISA reader at 630 nm.  Tools and Techniques to Study Apoptosis  Apoptosis, defined as programmed cell death, plays a very important role in many physiological and pathological conditions such as embryo and organ development, immune responses, tumor development and growth.  Detecting apoptotic cells or monitoring the cells progressing to apoptosis is an essential step in basic research and in developing drugs that may regulate apoptosis.  Apoptosis is characterized by many biological and morphological changes; such as, change of mitochondrial membrane potential, activation of caspases, DNA fragmentation, membrane blebbing and formation of apoptotic bodies.  Based on these changes, various assays are designed to detect or quantitate apoptotic cells. Typical assays include Annexin-V binding, DNA fragmentation and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling)  The detection of Annexin V-FITC by flow cytometry has become a hallmark for the separation of necrotic cells from those undergoing true apoptosis. Apoptosis Detection Method 1: Annexin-V The Annexin-V binding assay is based on the relocation of phosphatidylserine to the outer cell membrane. Viable cells maintain an asymmetric distribution of different phospholipids between the inner and outer leaflets of the plasma membrane. Choline-containing phospholipids such as phosphatidylcholine and sphingomyelin are primarily located on the outer leaflet of viable cells and aminophospholipids such as phosphatidylethanolamine and phosphatidylserine (PS) are found at the cytoplasmic (inner) face of viable cells. The distribution of phospholipids in the plasma membrane changes during apoptosis. In particular, PS relocates from the cytoplasmic face to the outer leaflet so called PS exposure. The extent of PS exposure can distinguish apoptotic cells from the non-apoptotic cells. Annexin-V is a 35-36 kDa calcium-dependent phospholipid binding protein with high affinity for PS (kDa ~ 5x10-10 M). When labeled with a fluorescent dye, Annexin-V can be used as a sensitive probe for PS exposure on the outer leaflet of the cell membrane. The binding of Annexin-V conjugates such as Annexin-V FITC to cells permits differentiation of apoptotic cells (Annexin-V positive) from non-apoptotic cells (Annexin-V negative).  Annexin-V binding is observed under two conditions:  The first condition is observed in cells midway through the apoptosis pathway. Phosphatidylserine translocates to the outer leaflet of the cell membrane.  The second condition is observed in very late apoptosis or when the cells become necrotic and membrane permeabilization occurs. This membrane permeabilization allows Annexin-V to enter cells and bind to phosphatidylserine on the cytoplasmic face of the membrane. Since other causes besides apoptosis can result in necrosis, it is important to distinguish between necrotic and apoptotic cells. Membrane permeabilization also permits entry of other materials to the interior of the cell, including the fluorescent DNA-binding dye propidium iodide. PI is impermeable and excluded from living cells. Cells that are stained negative for FITC Annexin V and negative for PI are considered living cells. Cells that are stained positive for both FITC Annexin V and PI are either in the end stage of apoptosis, are undergoing necrosis, or are already dead. Apoptotic populations can be distinguished from necrotic populations using the Annexin V-propidium iodide (PI) double staining regime - Non-apoptotic cells: Annexin-V negative and PI negative; - Early apoptotic cells: Annexin-V positive and PI negative; - Necrotic cells or late apoptotic cells: Annexin-V positive and PI positive. In summary, Annexin-V FITC/PI dual staining is very useful for defining apoptotic suspension cells by flow cytometry.  A. Cells were incubated with a certain drug that induce apoptosis and stained by Annexin-V FITC. Apoptotic and non-apoptotic cells can be differentiated according to Annexin- V FITC binding. M1: represents non-apoptotic cells and M2 represents Annexin-V positive cells. B. Staining with propidium iodide differentiates the Annexin-V positive population (apoptosis) from cells in necrosis. Conclusion: propidium iodide stained cells are easily distinguished from early apoptotic population (Annexin-V positive and PI-negative) appearing in the lower right quadrant of the two color dot plot. Apoptosis Detection Method 2: DNA Fragmentation Purpose: The Apoptotic DNA Ladder Detection Kit is designed for preparation of nucleic acids from mammalian cells to determine the level of DNA fragmentation of apoptotic cells. Principle of the Method Internucleosomal DNA fragmentation is considered a hallmark of apoptosis. During apoptosis, activated nucleases degrade the higher order chromatin structure of DNA into fragments of 50 to 300 kilobases and subsequently into small DNA pieces of about 200 base pairs in length. These DNA fragments can be extracted from cells and visualized by horizontal gel electrophoresis followed by ethidium bromide staining. The detection of DNA fragments by gel electrophoresis is one method to identify cells undergoing apoptosis. The Apoptotic DNA Ladder Detection Kit provides a simple and rapid procedure for extraction of chromosomal DNA. The procedure prepares DNA for use in the methods that detect DNA fragmentation in apoptotic cells. Unlike other methods which require 1 to 2 days to finish, this detection method only requires less than 90 minutes to prepare DNA in a single tube, without the need for extractions or column steps. DNA fragmentation can be easily visualized by agarose gel electrophoresis. This procedure increases recovery of small fragmented DNA and, therefore, improves the sensitivity of the assay.  Apoptosis Detection Method 3: TUNEL Detection Cell death by apoptosis is characterized by DNA fragmentation in 200-250 and/or 30-50 kilobases. Further internucleosomal DNA fragmentation in 180-200 base pairs may also occur. Such characteristics have been used to distinguish apoptotic cells from normal or necrotic cells. To detect apoptotic cells, whatever the pattern of DNA fragmentation, the TUNEL (Terminal deoxynucleotidyl transferase (TdT) mediated dUTP Nick End Labeling) method is commonly utilized. In the TUNEL assay (Apo-BrDU) exogenous TdT is used to catalyze a template-independent addition of bromodeoxyuridine triphosphates (Br-dUTP) to the free 3’-hydroxyl ends of double or single stranded DNA fragments. After incorporation, the labeled BrDU can be identified by FITC conjugated anti-Bromodeoxyuridine (BrDU) antibodies and analyzed using a flow cytometer or a fluorescence microscope. Due to the many free 3’-hydroxyl ends of fragmented DNA in apoptotic cells, a good signal is generated in affected cell populations. These cells can be visualized in tissue sections or quantified with flow cytometry. Compared to a single-step labeling FITC conjugated dUTP, this two step method provides a more sensitive, stronger signal.