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| 6.0µm Yellow <BR> | | 6.0µm Yellow <BR> |
| Polybeads are packaged as 2.5% aqueous suspension (water) and will be diluted to a working concentration of 0.08% prior to lab. This concentration approximately equals the working concentration of carbon particles in 1% India ink. <BR><BR> | | Polybeads are packaged as 2.5% aqueous suspension (water) and will be diluted to a working concentration of 0.08% prior to lab. This concentration approximately equals the working concentration of carbon particles in 1% India ink. <BR><BR> |
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| '''SAMPLE PROTOCOL TO INVESTIGATE THE ROLE OF ACTIN OR TUBULIN IN PHAGOCYTOSIS (you should modify/expand this for your experiments)'''<BR><BR>
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| #In a microfuge tube, make a 1:100 dilution of the drug inhibitor in ''Tetrahymena'' stock by combining 99 µl ''Tetrahymena'' (grown for 48hrs in 2% proteose-peptone) and 1 µl inhibitor. (Do not add more volume of inhibitor because some of the inhibitors are diluted in toxic compounds that could negatively affect the viability of your cells at higher concentrations.) Include an appropriate control for your experiment to make sure that your inhibitor's diluent is not affecting cell viability or phagocytosis. For example, if your inhibitor is dissolved in DMSO, your control should be 1 µl of DMSO added to 99 µl of ''Tetrahymena''.
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| #Incubate for 0-10 minutes if you think your inhibitor needs time to affect the element (actin, tubulin, or whatever you are trying to assess the role of in phagocytosis). Note that you can omit this pre-incubation step or shorten it if your research indicates that your inhibitor acts immediately on the element you are testing. What is the effective concentration of inhibitor in this important step?
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| #Add 100 µl of 1% India ink to the 100 µl ''Tetrahymena'' mixture prepared in step 1 in a microfuge tube. You will probably want to remove a 20 microliter sample immediately in order to assess a "zero time" control (see step 4). Calculate the concentration of your inhibitor and India ink after this combination. Note that you are making another ~1:100 dilution of your inhibitor in this step.
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| #At several time points ( ex. 0, 15, 30 min), REMOVE a 20 µl aliquot from the reaction microfuge tube of ''Tetrahymena''/inhibitor/ink and the control tube of ''Tetrahymena''/diluent/ink and, '''IN THE HOOD''', put each aliquot in a clean, labeled microfuge tube containing 10 µl of 3% glutaraldehyde. This will stop phagocytosis by fixing the cells so you can study them later. (If you would prefer to study your cells live, you will have to take your measurements very quickly to keep the timing relatively accurate.) What is the effective concentration of the gluteraldehyde fixative?
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| #'''IN THE HOOD''', make a slide for each time point by placing 20 µl of each tube from step 4 on a separate slide and cover each with a cover slip. Don’t make all your slides at the same time or the ''Tetrahymena'' will dry out before you can count the filled vacuoles.
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| #Count or assess whatever factor you have decided to measure in a reasonable number of cells at each time point. <br><br>
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| '''SAMPLE PROTOCOL TO INVESTIGATE THE ROLE OF PARTICLE SIZE IN PHAGOCYTOSIS (you should modify/expand this for your experiments)'''<BR>
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| There are many different approaches you might take to determine the affect of particle size on phagocytosis. You might want to determine the ability of ''Tetrahymena'' to phagocytose different particle sizes individually. For example, you might test the ability of ''Tetrahymena'' to phagocytose the 1.0µm blue, 3.0µm blue, 6.0µm blue, and 10.0µm blue Polybead dyed microspheres by exposing the ''Tetrahymena'' to just one of these blue microspheres at a time. Alternatively, you might be interested to know whether ''Tetrahymena'' are selective for particle size when exposed to microspheres of two different sizes at once. To address this question, you might expose ''Tetrahymena'' to the 3.0µm blue and 6.0µm yellow microspheres at the same time. Compared to counting ink filled vacuoles, it is more difficult to count the number of microsphere filled vacuoles (especially if you use the larger sizes). Therefore, you might design an experiment in which you quantitate the percentage of ''Tetrahymena'' that phagocytose beads of a particular size (as opposed to counting the number of filled vacuoles after exposure to different bead sizes). Alternatively, you might try to count filled vacuoles. A basic protocol is presented below.
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| #Add 40 µl of Tetrahymena to 40 µl of each size 0.08% Polybead dyed microsphere. Calculate the concentration of the polystyrene beads after this combination. (If you want to add 2 different colored microspheres at once, you need to determine if you want to keep the total microsphere concentration constant or if you want to keep a single microsphere concentration constant.) You will probably want to remove a 20 microliter sample immediately in order to assess a "zero time" control (see step 2).
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| #At several time points (ex. 0, 15, 30 min.), REMOVE a 20 µl aliquot from the reaction microfuge tube of Tetrahymena/beads and, '''IN THE HOOD''', put those aliquots in a clean, labeled microfuge tube containing 10 µl of 3% gluteraldehyde. (If you would prefer to study your cells live, you will have to take your measurements very quickly to keep the timing relatively accurate.) What is the effective concentration of the gluteraldehyde fixative?
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| #'''IN THE HOOD''', make a slide for each time point by placing 20 µl of each tube from step 2 on separate slides and cover each with a cover slip. Don’t make all your slides at the same time or they will dry out before you can count them.
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| #Count or assess whatever factor you decided to measure in a reasonable number of cells at each time point. <br><br>
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| Don't worry that you may not be able to measure everything you would like to measure because of limited resources, equipment, and time available. Simplicity in your experimental design usually makes execution less difficult and the results more reproducible.<BR><BR>
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| == Laboratory Cleanup == | | == Laboratory Cleanup == |
