Valerie AlcantarM1WS1.Separation of most blood cells is difficult, if not impossible, to achieve because they have similar properties and/or densities. What procedure is used to separate T-cells of the immune system from the many other different types of white blood cells or spleen cells? What feature of the T-cell facilitates the isolation protocol? Describe how this procedure is performed. T cells carry proteins CD3 and Thy 1.2 on their surface. When antibodies that are linked to fluorescent dye and specific for these proteins are incubated with a mix of cells, only the T cells will illuminate because they express the specific proteins and are recognized by the antibodies that carry dye. A fluorescence activated cell sorter which is based on flow cytometry can separate cells that emit different levels of fluorescence. A suspension of fluorescent and non-fluorescent cells are mixed with a buffer so that they pass in a single file through the laser beam. The fluorescent light of cell is measured as well as the laser light that is scattered by each cell which can determine the size and shape of the cell. The suspension then goes through a nozzle that forms tiny droplets containing a single cell. As each droplet that contains a cell is made, it is given a negative electric charge proportional to its fluorescence that was previously measured. The droplets then pass through an electric field and those with no cell thus no charge are removed and those with varying charges are separated and sorted. 2. Compare and contrast between scanning electron microscopy and transmission electron microscopy. In both styles of microscopy, electrons are extracted by a filament, accelerated by an electric field, and use condenser lens to focus on the specimen. In TEM, once the electrons pass through the thin slices of specimen they are focused by magnetic and projector lens to create a magnified image on a detector. In SEM, condenser lens along with objective lens are used to focus on a metal-coated specimen. Scanning coils are used to move the beam across the 3D surface of the specimen and the electrons that scatter from the metal are collected by a tube detector. Both microscopes also require the columns to be maintained at a high vacuum. 3. Rough endoplasmic reticulum can be separated from smooth endoplasmic reticulum by differential centrifugation. What is the basis for this fractionation? Once the cell is isolated, it will be disrupted to release its contents by suspending it in a solution that has upholds the condition that existed inside the cell. The first speed the homogenate is centrifuged is a low speed to remove the cell debris. Depending on what organelle is being isolated, the time and speed of centrifugation varies. The pellets formed at the bottom are the desired organelle or mixture of organelles. To remove ribosomes, nucleus, and microsomes from ER organelle, the speed of second centrifugation should be around 100,000 g. The liquid at the top is supernatant and contains organelles that require higher speeds to be isolated into a pellet. Because RER have ribosomes and SER do not, RER will require a higher speed. The fraction obtained from differential centrifugation can be purified by separating components according to their density called equilibrium density. Because RER have ribosomes they will be more dense than SER. 4. Describe how fura-2 is used to measure levels of intracellular Calcium ions. Fura-2 has five carbonyl groups that form ester linkages. It is able to diffuse across membranes into cells where esterases hydrolyze the fura-2 ester producing fura-2 molecule. In this state it has free carboxylate groups making it unable to cross cellular membranes. In the cell, it can only bind to a single calcium ion and nothing else thus its concentration is proportional to the concentration of calcium in the cytosol. This binding increases the fluorescence of fura-2 and does not change in different wavelengths nor does it change if calcium is not longer bound. With this, the rapid changes in the ratio in the amount of fura-2 fluorescence between two wavelengths can be observed thus knowing the calcium concentration in cytosol. 5. In certain electron microscopy methods, the specimen is not directly imaged. How do these methods provide information about cellular structures, and what type of structures do they visualize? TEM utilizes thin slices of the specimen and provides a 2 dimensional picture. TEM is based on transmitted electron versus scattered and it also has higher resolution. TEM can show morphology, internal composition such as lattice structures and particles crystallinity, can see objects as small as proteins. 6. Fixatives such as formaldehyde are routinely used in certain types of electron microscopy and light microscopy. However, fixatives may introduce complications in analysis of the resulting images. What problems may result from using fixatives? Part of fixation process requires dehydration that can alter the normal structure such as shrinking it. It can also alter the spatial relationship of cellular components.