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Lab-1 Studying Death at Cell level Behzad Khoshnood, 2015

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Cell death in multi-cellular eukaryotes Cell death Necrosis

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Development Homeostasis Immune survelliance Why is apoptosis beneficial for the organism? Apoptosis and cell proliferation must balance each other 5 X 10 11 blood cells are eliminated by apoptosis every day Killing of virus infected cells between the developing fingers

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Cellular morphology changes during apoptosis Normal cell Different stages of apoptosis Cell shrinkage Membrane ruffling DNA condensation DNA fragmentation Phagocytic cells will recognize phosphatidyl serine on the surface of the apoptotic cell and engulf and degrade it

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Apoptosis: Pathways Death Ligands Effector Caspase 3 Death Receptors Initiator Caspase 8 PCD DNA damage & p53 Mitochondria/ Cytochrome C Initiator Caspase 9

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The Fas/CD95/Apo-1 system in health and disease Fas Worn-out cell Virus infected cell Tumor cell Cytotoxic T cell carrying the Fas ligand on its surface Target cell killed through apoptosis upon cell-cell binding Fas FAS ligand

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JURKAT CELLS Established from a peripheral blood of a 14 year old boy with T cell leukemia immortalized line of T lymphocyte cells that are used to study acute T cell leukemia, T cell signaling, and the expression of various chemokine receptorsimmortalized lineT lymphocyteleukemiaT cell signalingchemokine receptors Easy to handle, robust growth(generation time~24hours) Presence of Fas receptor on the surface. Interaction of Fas ligand/Fas antibody to the receptor induces apoptosis

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Fas Apoptosis Fas antibody, activates the Fas receptor Your assignment To activate Fas-induced apoptosis using Fas antibody PMA PKC Apoptosis CD3 Prolifration

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Flow Cytometry Staining Western Blot (apoptotically-expressed proteins) TUNEL assay (fluorescent microscopy) Luminescent and Fluorescent Assays for Measuring Caspase Activity Microtiter plate assay COMMON METHODS FOR MEASURING APOPTOSIS Apoptotic cells Normal cells

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1mm 2 Counting cells using Trypan Blue Harvest cells Re-suspend cells in fresh medium Take 25l of cell suspension & Mix equal volume of Trypan Blue Load the chamber with cell suspension (10l) Count & Calculate avg number of cells Total number of cells [cells/l] = Average number of cells counted/(counted area [mm sq.] chamber depth [mm] dilution factor) Ex: Mix 100 l cells with 100 l Tryphan Blue and inject 10 l under the coverslide Count all live cells in 3 squares and get the avg no of cells. Square area:1mm 2 Chamber depth: 0,1 mm Dilution factor: First calculate the number of cells per square: total number/3 squares = avg number This give an equation like: Avg cell count(1mm 2 0.1mm 1/2) = no. of cells / l => no. of cells / ml

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Calculation of cell densities using a Brker chamber 1 A-square (9 A-squares in total) 1.Mix 20l Cell culture with 20l Trypan Blue. 2. Carefully pipette solution onto the chamber until it is filled (~15l). 3. Count the mean number of cells per A-square (of at least 5 squares) 4. Calculate cell density: (Mean cell number per A-square) * 2* 10.000 = Number of cells / ml

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~0.1 x 10 6 cells on a glass slide Label Funnel Filter card Glass slide Slide holder Cell suspension, 100 - 200 l 500 rpm during 2 minutes Preparation of cells for microscopy

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Staining of cells for microscopy 1.After cytospinning let glass slides dry briefly (10-15 min) 2.Stain slides for 5 minutes in May-Grunwald solution 3. Stain slides for 20 minutes in Giemsa solution 4. Wash slides 3 X 5 minutes in water 5. Let slides dry, thereafter they are ready for observation in your light microscope (x40 magnification) in Cytosol (May-Grunwald) Nucleus (Giemsa)

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Apoptotic cell Healthy cell How do they look like?!