Difference between revisions of "Optical Microscopy Part 4: Particle Tracking"
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You are provided with NIH 3T3 fibroblasts, which were prepared as follows: | You are provided with NIH 3T3 fibroblasts, which were prepared as follows: | ||
Cells were cultured at 37°C in 5% CO<math>_2</math> in standard T75 flasks in a medium referred to as DMEM++. This consists of Dulbecco's Modified Eagle Medium (DMEM - Invitrogen) supplemented with 10% fetal bovine serum (FBS - Invitrogen) and 1% of the antibiotic penicillin-streptomycin (Invitrogen). The day prior to the microrheology experiments, fibroblasts were plated on 35 mm glass-bottom | Cells were cultured at 37°C in 5% CO<math>_2</math> in standard T75 flasks in a medium referred to as DMEM++. This consists of Dulbecco's Modified Eagle Medium (DMEM - Invitrogen) supplemented with 10% fetal bovine serum (FBS - Invitrogen) and 1% of the antibiotic penicillin-streptomycin (Invitrogen). The day prior to the microrheology experiments, fibroblasts were plated on 35 mm glass-bottom | ||
| − | cell culture dishes (MatTek). On the day of the experiments, the cell confluency should reach about 60%. 1 μm diameter | + | cell culture dishes (MatTek). On the day of the experiments, the cell confluency should reach about 60%. 1 μm diameter red fluorescent microspheres (Corpuscular) were mixed with the growth medium (at a concentration of 5 x 10<math>^5</math> beads/mL) and added to the plated cells for a period of 12 to 24 hours for bead endocytosis. |
# Carefully pipet out the medium from both dishes. Replace with 2 mL of fresh, pre-warmed DMEM++ in each dish. | # Carefully pipet out the medium from both dishes. Replace with 2 mL of fresh, pre-warmed DMEM++ in each dish. | ||
Revision as of 13:55, 4 September 2013
In this part of the lab, you will investigate how a drug called cytochalasin D (CytoD) affects the structure and rheological properties of fibroblast cells. Immunofluorescent staining will be used to make the actin cytoskeleton of fibroblast cells visible. You will compare the structure of cells that have been treated with CytoD with that of untreated cells. In the next part of the procedure, you will measure how CytoD treatment affects the microrheological properties of cells. You will track small, fluorescent microspheres inside of the cells for several minutes. Using the particle tracks, you will calculate the frequency-dependent storage and loss moduli of the environment inside the cells.
Cell culture and labeling protocols
Read Optical Microscopy Part 3c: Protocols for cell culture, cell fixing and cell imaging.
Instructions
Actin imaging
Actin can be imaged using immunofluorescent staining with a mushroom toxin called phalloidin. The phalloidin you will use has been conjugated to a fluorescent dye called Alexa Fluor 568. Phalloidin binds to polymerized filamentous actin (F-actin), but not to actin monomers (G-actin).
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Wear gloves when you are handling biological samples. |
Since actin filaments and stress fibers are nanometer-scale objects, they are much dimmer than fluorescent
beads or the dye solution - care must be taken to get good images of the cytoskeleton. You may
need to cover the microscope to reduce room light contamination.
- Adjust the gain and exposure of the camera to get the best picture. Be sure to keep the same exposure conditions, however, for both untreated and CytoD-treated cells.
- Using the 40x objective, take some good fluorescence images of Alexa Fluor phalloidin-labeled cells, both untreated and CytoD-treated.
Live cell microrheology by particle traking
Now that you have verified microscope stability and performance through the particle tracking lab, you can apply its methods on cell samples. A key technique to keep in mind when working with live cells - to avoid shocking them with "cold" at 20°C - is to be sure that any solutions you add are pre-warmed to 37°C. We will keep a warm-water bath running for this purpose, in which we will keep the various media.
You are provided with NIH 3T3 fibroblasts, which were prepared as follows: Cells were cultured at 37°C in 5% CO$ _2 $ in standard T75 flasks in a medium referred to as DMEM++. This consists of Dulbecco's Modified Eagle Medium (DMEM - Invitrogen) supplemented with 10% fetal bovine serum (FBS - Invitrogen) and 1% of the antibiotic penicillin-streptomycin (Invitrogen). The day prior to the microrheology experiments, fibroblasts were plated on 35 mm glass-bottom cell culture dishes (MatTek). On the day of the experiments, the cell confluency should reach about 60%. 1 μm diameter red fluorescent microspheres (Corpuscular) were mixed with the growth medium (at a concentration of 5 x 10$ ^5 $ beads/mL) and added to the plated cells for a period of 12 to 24 hours for bead endocytosis.
- Carefully pipet out the medium from both dishes. Replace with 2 mL of fresh, pre-warmed DMEM++ in each dish.
- Choose cells with 3 or 4 particles embedded in them and capture movies of the samples. Take multiple movies with about 3-5 cells in the field of view in each movie.
- In one of the dishes, treat the cell with the cytoskeleton-modifying chemical cytochalasin D (CytoD): Pipet out the medium, add 1 mL CytoD solution at 10 μM (pre-mixed for you) to the dish, and wait for 20-30 minutes. It's a good idea to check on your cells after 20 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 as quickly as you are able. The cells' physiology has now been significantly disrupted by the toxin CytoD, and they will die within a couple of hours. It's very unlikely that you'll be able to find the exact same cells you've already tracked; however it's very much advisable to use the same dish for the "before" and "after" so you're aren't also comparing between different cell populations.
Lab manual homepage
Lab Manual: Optical Microscopy
References
