Experiment 7: Phagocytosis
Dictyostelium amoebae eat bacteria as their normal food source. However, bacteria are too small to easily visualize their uptake in the light microscope. Instead, we use yeast cells that have been killed by treating them with ethanol. For some reason, live yeast are not eaten. Phagocytosis is an actin driven process, so using a GFP probe that binds to actin filaments (GFP-filABD) you can visualize and monitor the process of phagocytosis. The yeast are also expressing a red fluorescent protein. It may be possible to measure the degradation of the fluorescent protein by lysosomal proteases after uptake, but we have not tried this yet and we don’t know how long it would take.
There are many possible variations on the theme of this experiment, and you can explore several additional aspects of this topic in a variety of ways:
1. Mike has recently discovered that adding vitamin C to media dramatically improves the resistance of cells to the toxic effects of the fluorescent light. Vitamin C is an antioxidant and it apparently removes photooxidation products before they can damage the cells. He will provide you with a concentrated stock of vitamin C (xx mg/ml which is xx fold concentrated). This has not yet been tested on the LED illuminators, so you can try this by taking images fairly frequently so the cells round up, add the ascorbic acid and then try again. We don’t know how long it will take to have an effect on the cells, but there are hints that you might have to treat the cells with vitamin C for as much as 3 hours before it has full effect. We also don’t know the optimal concentration so that is another parameter that could be explored. This all can be looked at in the context of phagocytosis specifically, or optimized just looking at the GFP-filamin probe in vegetative cells crawling and endocytosing before you do it with yeast.
2. For phagocytosis, I would try this first with a standard plastic petri dish at 20 or 40x to visualize the speed and frequency of phagocytosis events. It should happen fairly rapidly, so don’t add yeast until you are ready to image.
3.You can use ibidi dishes if you wish to go to 100x oil immersion to image the phagocytosis process
4.We also have 8 well chamber slides that have two rows of 4 chambers which may be convenient for testing a variety of parameters. The bottom of the dish is a coverslip, so they are designed for oil immersion microscopy. Each small chamber holds about 250 µl of media so you can set up 8 chambers of cells and use them sequentially. However, it is really hard to use the outside wells because they are close to the tabs that support the chamber on the stage. So I recommend only using the inner 4 wells of a chamber. If you use them, they should be cleaned when you are done. Ask Mike or I how to gently clean them for reuse.
5.Once you have imaged the uptake process, you can switch to the green illuminator and see if you can image the degradation process. This will happen on a much longer time scale than uptake, but as indicated above, we don’t know how long.
Basic protocol for initiating phagocytosis
1)Plate 2x106 cells (this seems high to me- is it correct?) in a p30 dish and allow them to settle and attach for at least 20 minutes.
2)Once the cells have settled, gently remove the HL5 media with a pipette and replace the media with MCPB (with or without ascorbic acid).
3)Put the plate of cells onto the microscope and find the correct focal plane. Once the focal plane is obtained, set up the timelapse using the blue and white channels, taking images every 5-10 seconds. Once you are ready, it is a good idea to move to a new field of view at this point so that your cells have not been exposed to light continuously. You can do all this with the lid off the dish to make it easy to add the yeast. Evaporation is not a concern in the short time of this experiment.
4)Start the time lapse running
5)When you are happy with how the cells are behaving, measure out 50 µl (based upon the surface area of a P30) of the concentrated yeast solution (made up in MCPB).
6)Hold the pipettman over the dish and GENTLY and slowly expel the yeast out of the pipette tip until it forms a droplet at the end of the tip.
7)Touch the yeast droplet onto the media in the Dictyostelium plate directly over the objective.
8)You will slowly begin to see yeast particles settling into the focal plane and the Dictyostelium cells will begin the process of phagocytosis almost immediately.
