Bradford Assay of proteins Coomassie Blue dye exhibits a spectral shift upon binding various side chains Within certain concentration ranges protein concentration is directly proportional to the concentration of blue No bubbles o Bubbles effect the spectral reading Microplate reader to analyze Trypan Blue Dye Exclusion Assay Assay for plasma membrane permeability cell viability Apoptotic cells have permeable membranes o Stain blue Wright Stain Assay Assay for nuclear fragmentation Granulocyte nuclei stain dark blue purple Dye made up of methylene blue and eosin dyes in methanol and observed under compound light microscope Allows us to detect nuclear fragmentation Apoptosis Annexin V FITC Propidium Iodide Fluorescence Assay for plasma membrane asymmetry and permeability Annexin V FITC binds to PS and fluoresces green Propidium Iodid enters cytoplasm and fluoresces red o Excited with blue light o Excited with green light WST 1Assay Colorimetric assay for cell viability Absorbance of formazan product analyzed in a microplate reader Quantify viable cells in sample Viable cells possess active oxidoreductase enxymes that are capable of reducing tetrazolium dye to the colorimetric product formazan Calculates absorbance in the sample Absorbance of formazan is directly proportional to the number to the number of metabolically active cells in the sample No color product cells dying Caspase Assay Colorimetric assay for caspase activity Absorbance of pNA product analyzed in a microplate reader Caspase Cascade triggered by cleavage of pNA Cleavage of protein substrates causing the disassembly of the cell Genomic DNA Isolaion and Agarose Gel Electrophoresis Assay for DNA fragmentation property of apoptotic cells Fragmented DNA is separated by number of base pairs in agarose gel electrophoresis DNA fragmentation a feature of apoptosis Microscopy Likely questions Bright field compound light microscope or phase contrast microscope Phase contrast condenser ring o Phase shifts between diffracted and undiffracted light are exaggerated higher contrast Measure with ruler on microscope o Ocular micrometer o Ocular scale small close together dashes o Stage micrometer scale bigger spaced out 0 1mm 100um How many microns per ocular unit 100um 10oc u 10um per ocular unit See sheet attached Fluorescent microscopy o Blue excitation emits green o Green excitation emits red different methods of cell death main caspases Main types cell death o Apoptosis Death by suicide internal signals external signals o Necrosis Death by injury Mechanical damage Exposure to toxic chemicals o Cellular changes of apoptosis Mitochondrial Recruitment specific proteins to mitochondrial membrane Release of cytochrome c Changes in mitochondrial membrane potential Plasma membrane Change in phospholipid distribution Late changes in membrane permeability Enzymatic changes Cleavage of caspase enzymes Proteolytic cleavage of their substrates The caspases Caspase 3 activity o DNA Fragmentation o Intrinsic or extrinsic both can activate Caspase 8 death receptors o extrinsic Caspase 9 o Triggers apoptosis in response to extracellular factor that activates o Activation is triggered by release of cytochrome c in mitochondria during intrinsic pathway o Mitochondrial stress o Chytochrome c released then 9 activvated o APOPTOSOMES o Intrinsic Antagonist Agonist Slows heart rate bpm Endogenous Ligand binds to and activates receptor causing cellular response or agonist binds and activates the same receptor causing cellular response that will vary Increases heart rate bpm Antagonist binds but the interaction does NOT activate the receptor As a result there is no cellular response to binding What ion does what See attached Facts about the lab Heart muscles cells cardiac myocytes spontaneously contract without stimulation by nerve cells o Contraction caused by propagation of action potentials originally generated in a specific group of myocytes Called pacemaker cells Clustered in region of the R atrium of the heart called the Sinoatrial node Spontaneously depolarize spreads to neighboring cells via gap junctions Rate and strength of contraction regulated by sympathetic and parasympathetic nerves of the autonomic nervous system Repolarization and depolarization study the curve of this likely a question because we discussed it in class a lot The standard curves and dilution factors Will probably ask questions on this so understand how to make the curve and how to gather information from it
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