The overall goal of this procedure is to measure the flow of cytoplasm during wound healing in a single cell. This is accomplished by first collecting live stent or SEIUs cells to use as a model system for cellular wounding. The second step is to prepare the glass tools for cutting the cells.
Next, the stent sarius cells are cut open on a customized microscope setup that allows for simultaneous low magnification surgery and high resolution imaging. The final step is to perform computational analysis of the cytoplasmic flow using particle image velo symmetry software. Ultimately, high resolution imaging of the cytoplasm is used to show the active response of cells during wound healing.
This method can be used to address key questions about cellular wound healing, such as whether or not the cytoplasm becomes gel-like around the region of a cut in order to prevent leakage into the surrounding medium. Visual demonstration of this method is critical as the cell handling tool preparation and cutting steps are difficult to learn as they require manual dexterity. To begin obtain stent or Sirius cells from either natural pons as described by tartar or from a commercial supplier, add 300 milliliters of modified stent or medium adapted from recipes by tartar and deter as described in the accompanying text protocol.
Maintain cultures by growing them in the dark at 20 degrees Celsius. Next, grow up cultures of chlamydomonas Rhine Hardy algae in tris acetate phosphate medium. Using standard culture methods prior to feeding, wash the chlamydomonas cells by gently spinning them down, removing the supernatant and suspending them in the modified stent or medium.
Feed the 300 milliliters stent or sarius cultures with about 30 million chm cells two or three times per week when needed. Collect stent or cells for surgery. To begin, gently agitate the culture by bubbling air through a pipette tip until the cells begin to detach and swim freely.
Then capture the swimming cells using a one milliliter pipette equipped with a wide bore tip to avoid shearing the cells. Once collected, let the cells settle. Then slowly pipette away the media above them and add fresh modified stent or medium.
Prepare the glass needles used to cut the stent cells by holding two glass stirring rods over abundance and burner until the ends begin to melt. Then touch the ends of the two rods together until the molten glass fuses. Once they fuse, remove them from the flame and allow them to cool for one to two seconds.
Then slowly pull them apart to make a clean break. Ideally, this will produce a needle that is approximately one to two centimeters in length with a fine point at the end. If the resulting needle is suboptimal, break the tip into the glass waist and reuse the rods.
Next, use a square rubber stamp coated in Vaseline to prepare a square Vaseline walled chamber by pressing it into a glass cover slip. Then add 100 microliters of modified stent or medium containing 2%methyl cellulose to the Vaseline square. Place the prepared cover slip into a plastic adapter and then add the washed stent cells to the cover slip.
Finally, arrange cells in the methyl cellulose so that they are evenly spaced and are pushed close enough to the surface of the cover slip to be in focus during imaging. To begin, place the slide onto the stage of the inverted microscope. Then remove the dissecting microscope body from its stand and determine the approximate position in which it will need to be placed in order to visualize the slide at low magnification from the top.
Next, construct a double decker microscope by adding a cardboard box with the desired spacing between the eyepiece of the inverted microscope and the dissecting scope. Tape the box into place using lab tape so that it can be easily removed When the experiment is completed, make sure that the sample will be in the field of view and in focus without needing to move the dissecting scope After it's attached, then hold the dissecting scope against the box and tape it onto the box using lab tape. Next, set up the inverted microscope using DIC optics by putting a 20 x air objective into place.
Also, make sure to use a long working distance DIC condenser to provide enough space to perform the microsurgery from above. Then bring the cells into focus with the inverted microscope with everything prepared and in focus from both sides. Begin to acquire images at a frame rate of two images per second, using a five 12 by five 12 CCD camera.
Then cut the cell using the glass needle. Capture the initial cytoplasmic flow pattern and observe all stages of the wounding and wound healing process. Terminate the acquisition when the cell has resumed normal appearance or has once again begun active motility.
To begin download PIV lab a MATLAB package to measure cytoplasmic flow using particle image veloc symmetry. Next, load the TIFF image stack for each video sequence into the particle image veloc symmetry program Within MATLAB manual. Manually generate a mask for each image stack to mask out regions of the raw data outside the body of the stent.
Then set PIV lab to use the fast Fourier transform setting and a bin size of 64 by 64 pixels, which will bin the pixels in each frame into larger square pixels. Next run PIV lab to compute cross correlations between the pixels in each frame with nearby pixels. In the next frame, this will yield a time dependent flow vector centered at each bend pixel, which has the same time resolution as the raw data.
Then compute time averages of the resulting vector field output in order to compare the data taken with the stent before cutting and during wound healing. Also, calculate histograms of flow velocities to study flow strength and change of velocity at a given pixel over time in order to study flow fluctuations. When imaging sequences with the stent or cell cleaved in half, calculate the total flux of cytoplasm back into the cell body proper by multiplying the average flow velocity by the cross-sectional area of the extracellular cytoplasm bubble base.
Shown here is live imaging of a stent or cell as it is cut and disrupted with a glass needle. After cutting, a substantial quantity of cytoplasm has been pulled out of the main cell body, but it rapidly flows back into the cell. Following this procedure, chemical inhibitors may be added in order to answer additional questions such as what cytoskeletal components might contribute to the cytoplasmic motion during stent or wound healing.
After watching this video, you should have a good understanding of how to prepare the tools for cutting stent to our cells and how to image cytoplasmic flow during the cutting and wound healing process within the single giant cell.
