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Lecture 3

Biology 2290F/G Lecture 3: Isolation and Analysis of Cell Organelles & Molecules

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Biology 2290F/G
Ray Zabulionis

Lecture 3: Isolation and Analysis of Cell Organelles & Molecules Labelling live cells with fluorescent antibodies or stains - Antibodies made against specific cell surface proteins can be linked to fluorophores - Membrane permeable fluorescent dyes can be used to label intracellular structures (e.g. Hoechst stain binds DNA in nucleus) - Cells with bound antibodies or that have taken up the dyes can now be sorted and counted - Say we want to characterize a certain type of white blood cells, which express a certain type of proteins - They're transmembrane proteins, with a bit that sticks out on the outside - They have these things called CD3 and Thy1 2 - We have an antibody that recognizes Thy1, and another one that recognizes CD3 - Antibodies can be conjugated to fluorophores, where they can recognizes proteins on the outside of the cell - We can add membrane permeable fluorescent dyes that can be used to label intracellular structures - We now have 3 different potential fluorescent parameters we can monitor - Thy 1 in cells will have green fluorescence, CD3 cells will have red fluorescence, and all nucleated cells will fluorescence blue Fluorescent Activated Cell Sorting (FACS) 1. Cells pass through single file through a laser light beam 2. Both fluorescent light emitted and scattered are measured by detectors 3. Individual cells are forced through a nozzle and given a charge proportional to the degree of fluorescence detected 4. Cells with different electric charges are separated by an electric field and collected - We have illumination sources (e.g. lasers) that are focused on a tube that transports fluid - In the fluid are the cells we've labelled - Allows individual cells to flow through - Laser light illuminates the cells that are passing through o Light is tuned to a specific wavelength which causes excitation of the fluorochromes that are labelling the samples o Light that's emitted can be detected with light detectors o One detector for each colour of light - Each cell that comes through can be recognized, and amount of fluorescence coming off the cell can be quantified - If we want to isolate cells that have high amounts of both red and green fluorescence, we can apply a high electric field to the cells, and the ones that have a high field applied to them will drop with a greater charge Quantification of cells expressing two different cell surface by FACS - As the cells pass through the FACs machine, the intensity of the green and red fluorescence emitted by each cell is recorded - Each dot represents a single cell - The proportion of each cell population can be calculated Cell Cycle Analysis by FACS - Cells that have replicated their DNA but not fully divided (G2) will have twice the Hoechst stain for fluorescence intensity of non-dividing cells (G1) - Graph shows the number of cells with a certain intensity of fluorescence - We can determine the cells in different stages of the cell cycle: before and after replication - In many cells, the longest phase of the cycle is G1 (before DNA synthesis) o This explains the high peak in the histogram o These cells have double the DNA because they've replicated the DNA, and it's before the cells have physically divided - Cells in the process of replicating DNA is the S stage - We can learn a lot about cell cycle by labelling cells with Hoechst stain and passing them through FACS o We can then quantify the amount of DNA in the cell, which is proportional to what the cell is doing in terms of the cell cycle o More DNA present, more fluorescence How to isolate cell organelles? Step one: disruption of cell plasma membrane - Mechanical homogenization - Sonication (ultrasound) - Pressure (cells forced through a very narrow valve) - Non-ionic detergents (e.g. Triton X-100) - Placing cells in hypotonic solution Step two: centrifugation of cell homogenate - Differential - Equilibrium density-gradient - Tube where we suspend cells, and put a pestle in the tube o Go up and down, and this will squish the cells between the pestle and side of the tube, and that mechanical restriction causes the cells to lyse - To disrupt all membranes, we can use sonication - Force cells through a narrow valve which will mechanically disrupt the cell - Treat cells with non-ionic detergents New and Used Centrifuges - Centrifuge goes at high speed - Important to balance the centrifuge; if you don't and it gets up to full speed, it can break off the axel and shoot through the wall of the chamber Differential Centrifugation - Spinning homogenate yields pellet and supernatant - Increasing centrifugal force (gravity) to isolate organelles based on mass 1. Filter homogenate to remove clumps of unbroken cells, connective tissue etc. – end up with your filtered homogenate 2. When isolating components of cell, we first do differential centrifugation 3. We have free floating mixture of nuclei and organelles 4. Largest structure will be the nuclei 5. When we centrifuge at about 600g, the supernatant contains everything that's not the nucleus, and the pellet at the bottom is the nucleus 6. Then we do 15 000g x 5 min, which will pellet the smaller organelles 7. Centrifuge a
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