This is Part 2 of Assignment 5.
Live cell particle tracking of endocytosed beads
We can also use particle tracking to probe cell samples. 0.84 μm diameter red fluorescent microspheres were mixed with the growth medium and added to the plated cells for a period of 12 to 24 hours for bead endocytosis.
You will be given two plates of cells for these experiments. The cells must be imaged while they are alive, so the cells must be used the day you are given them:
- Dish 1 will be used to monitor particles in untreated cells, while
- Dish 2 will be reserved to track microspheres after adding CytoD.
- Pre-warm your DMEM++ and CytoD to 37°C
- Carefully pipet out the medium from Dish 1. Gently rinse with 1mL of medium 2X to remove beads that were not endocytosed. Then, place 2 mL of fresh medium in dish.
- Choose cells in Dish 1 with at least 2 but preferably 3 or 4 particles embedded in them and capture movies of the samples. Make sure to do this quickly, as the cells become unhealthy without the temperature and carbon dioxide regulation.
- By adjusting the LED current and exposure time, you should be able to use both bright field and fluorescence illumination simultaneously to find cells containing enough beads. Once you find a good-looking cell, turn off the LED and readjust the exposure time appropriately.
- Take as many movies as you can with about 2-5 particles in the field of view in each movie.
- Next, carefully pipet out the medium in Dish 2. Gently rinse with 1mL of medium 2X to remove beads that were not endocytosed.
- Treat the cells in Dish 2 with the cytoskeleton-modifying CytoD: Pipet out remaining medium, add 1 mL pre-warmed CytoD solution at 10 μM (pre-mixed for you) to the dish, and incubate for 20 minutes at 37°C. It's a good idea to check on your cells after 15 minutes: sometimes they are in bad shape at that point but sometimes they still look very healthy. Wash 2X with 2 mL of pre-warmed DMEM++, leaving 2 mL in the dish when imaging.
- Perform and repeat the particle tracking measurements again in Dish 2 as quickly as you are able. It would be good to image the beads in only one cell at a time, since different cells may have different degrees of cytoskeletal disruption. Take as many videos as you can before the cells become sad. The cells' physiology has now been significantly disrupted by the toxin CytoD, and they will die within a couple of hours.
||Turn in for particle tracking in cells:
- Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)
- Include a snapshot of the 0.84 μm fluorescent beads monitored.
- Plot two or more example bead trajectories for each of the samples. (Hint: If you subtract the initial position from each trajectory, then you can plot multiple trajectories on a single set of axes.)
- Analysis and Results
- Combine your data with others from the class to increase your sample size
- Plot the average MSD (from the difference trajectories) for untreated and Cyto D treated cells on a single set of log-log axes.
- What kind of motion do you see described by your MSD vs τ results?
- What differences do you see between the untreated and Cyto D treated MSD curves?
- Please suggest an interpretation of the behavior of your cells based on your data.
- Include a discussion of your error sources.
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