The overall goal of this protocol is to grow Stentor for their use as a model organism to study regeneration at the single cell level. This method can help answer key questions in the cell biology field such as how do cells build structures from discrete parts. The main advantages of Stentor are that they're single cells that can regenerate and that they're big enough to perform surgery on.

Generally the challenging part of this protocol is that people are used to working with cells as cultures rather than as individuals. Begin by searching for Stentor sample-containing Petri dish under a stereo microscope using oblique light at a 5X magnification. Note that Stentor cells exhibit a trumpet-like shape when they are attached to a substrate.

While non-attached, swimming Stentor cells are less extended in appearance. Using a one milliliter pipette, transfer individual Stentor cells into the well of a glass spot plate containing at least 100 microliters of commercially available pasteurized spring water. Then remove about 90%of the water in the well without removing the Stentor cells and add 500 microliters of fresh pasteurized spring water to the well three times to wash the cells.

To feed the cells, first transfer a one milliliter aliquot from a Chlamydomonas culture into a 1.5 milliliter microcentrifuge tube for centrifugation at 2, 095 g for three minutes at room temperature. Resuspend the pellet in one milliliter of pasteurized spring water for a second centrifugation, followed by resuspension in 500 microliters of pasteurized spring water. If a clonal Stentor culture is needed, transfer 500 microliters of conditioned medium per culture to individual wells of a glass spot plate and transfer one Stentor cell into each well of medium.

Feed each Stentor culture five microliters of the prepared Chlamydomonas every 48 hours and keep the cultures in a shaded area. Count the number of new Stentor cells before each feeding and adjust the volume of delivered Chlamydomonas accordingly. Every 96 hours, gently pipette the culture medium a few times to detach the Stentor from the meniscus of each well and use a one milliliter pipette to carefully aspirate the media from the wells without removing any cells.

Then add 500 microliters of fresh conditioned media to each well. When the number of cells in at least one well exceeds 20, add 20 milliliters of pasteurized spring water to an autoclaved sterilized wide mouth glass jar and carefully detach the Stentor cells from the bottom of each well as demonstrated. Collect all of the Stentor cells with a one milliliter pipette and gently transfer the cells to the glass jar.

Add fresh pasteurized spring water to the jar every 48 hours to keep the Stentor density to about 20 cells per milliliter and feed the Stentor cells with 200 to 1, 000 microliters of prepared Chlamydomonas. When the culture volume reaches about 90%of the jar's capacity, transfer the entire contents of the jar into a two cup glass container feeding the high volume Stentor cultures two milliliters of prepared Chlamydomonas per 100 milliliters of culture and adding pasteurized spring water to the culture every four to five days to keep the Stentor density at about 20 cells per milliliter. Once a week, inspect the cultures under a 5X dissecting microscope for rotifers, fungus, and other growth using a one milliliter pipette to remove any contaminating microorganisms along with any abnormally shaped and colorless Stentor cells.

Split the culture when the glass container is about 90%full, adding 25 milliliters of pasteurized spring water to the culture and using a 50 milliliter pipette to detach the Stentor from the glass as demonstrated. Then move about 50%of the culture into a new two cup glass container. To induce regeneration by cell fragmentation, first use a needle puller to make several needles from capillary tubes.

Next, collect one healthy Stentor with a defined trumpet shape, vibrant blue-green color, and no large vacuoles in a two microliter droplet. Place the droplet on a slide and slow the motion of the Stentor with two microliters of 4%methyl cellulose. Place the droplet under a dissecting microscope and hold the glass needle as parallel to the cutting surface as possible to prevent needle breakage.

Gently press the contracted Stentor with the side of the needle to split the anterior end of the cell from the posterior end of the cell. Confirm that both of the fragments have at least one macronuclear node and move the fragments into individual wells of a glass spot plate. To induce membranellar band and oral apparatus regeneration, transfer 30 to 60 Stentor cells in one milliliter of culture medium into a microcentrifuge tube and use a 200 microliter pipette to collect all of the cells from the tube in a single 125 microliter volume draw.

Dispense the cells into a tube containing 500 microliters of freshly prepared 25%sucrose and start a stop watch. After flick spinning the cells in the tube in the rack for one minute, collect all of the cells in a single 200 microliter draw and hold the cells in the pipette tip until the stop watch displays two minutes of sucrose treatment. Then eject the Stentor into a microcentrifuge tube containing one milliliter of pasteurized spring water for three flick spin washes.

To image the regeneration, add 100 microliters of pasteurized spring water into one well of a glass spot plate and use a 20 microliter pipette tip to collect one Stentor cell in four microliters of culture media. Deposit the droplet into the middle of a 22 by 22 square millimeter coverslip and place four more droplets around the previous droplet. Invert the coverslip and gently place it over the well with pasteurized spring water.

Then image the regenerating cells under a light microscope with the appropriate time resolution. During stage one of regeneration, which occurs immediately after the sucrose washout, the Stentor cells look like teardrops without any membranellar band. After three to six hours, a membranellar band appears.

This stage, stage two, last another three to four hours. Stage three occurs when an oral primordium appears at the posterior end of the membranellar band and both structures start moving toward the anterior end of the cell. This stage lasts for one to two hours.

When both the membranellar band and the oral apparatus reach the anterior end of the cell, the regeneration is complete and the cell will have adopted the characteristic Stentor trumpet-like shape. Plot the percentage of cells in each regeneration stages at each time point in a stacked box plot. Measuring the regeneration time after cell cutting and sucrose treatment reveals an hour-long spread in the time taken by the population of cells to reach any particular stage demonstrating the temporal heterogeneity of the regeneration process within a given population of regenerating cells.

After watching this video, you should have a good understanding of how to culture Stentor and initiate their regeneration and quantify it. While attempting this procedure, it's important to remember to be patient. Stentor take three to five days to divide in culture and identifying the Stentor regeneration stages takes practice.

This technique will pave the way for cell biology researchers to explore regeneration in Stentor.

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