{'fraction_non_alphanumeric': 0.020429252782193957, 'fraction_numerical': 0.008028616852146264, 'mean_word_length': 4.773749426342359, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.


Introduction
Cells are not simple bags of enzymes, but rather highly complex machines whose components are carefully scaled to the correct size and arranged in well-defined positions. The morphogenesis of individual cells represents a key process in cell and developmental biology, but its molecular mechanism is unknown 1,2 . While some cultured cells resemble blobs, unicellular organisms can have extremely complicated architectures, exemplified by the complex cortical patterns seen in ciliates 3,4 . Perhaps the most extreme example of a highly structured cell is Stentor coeruleus, a giant heterotrichous ciliate distantly related to Tetrahymena and Paramecium. Stentor is 1 mm long and is covered with more than 100 longitudinal stripes of blue pigment alternating with rows of cilia organized by parallel stacks of microtubule ribbons that run the length of the entire cell. The cell is trumpet-shaped (Figure 1), with a membranellar band and an oral apparatus (OA) at its anterior end, and a holdfast that attaches the cell to the substrate at its posterior end. In addition to the clear anterior-posterior polarity, the cell also shows a distinctive chiral patterning, such that spacing between ciliary rows gradually increases in a clockwise direction. This results in a discontinuity where the narrowest row meets the widest row, and this region of the cell surface, known as the locus of stripe contrast, can induce the formation of the second set of anterior end structures when grafted onto another cell 5 , making it formally equivalent to Spemann's Organizer. Thus, all key processes of developmental biology have their analogs in Stentor: axiation, pattern formation, and induction. In an embryo, these processes are driven by fate differences between different cells, but in Stentor, they must be driven by fate differences between different regions within a single cell. What defines the differences between the regions within Stentor is a mystery.
If any part of Stentor is cut off, the missing piece of the cell can regenerate to yield a normal cell in a matter of hours. If a cell is cut in half, or even into much smaller pieces, each piece reorganizes into a normal-looking but smaller cell and restores proper proportionality between cell parts 6,7 . Even tiny fragments, 1/64 th the size of the original cell, are able to regenerate into a small but normally proportioned cell, and then grow to the full size 6 . Stentor thus presents a unique opportunity to study the mechanisms of organelle size scaling and cell growth regulation using surgical methods that are usually applied at the level of tissues or whole organisms.
One of the properties of Stentor that allows it to regenerate from a wide range of surgical operations is that it contains a single nodulated macronucleus (Figure 1) with about 50,000 copies of the entire genome . 1. To collect Stentor from a pond, lake, or river, find an area with some vegetation where the water is relatively calm, shady, and clear.
Note: There is a higher probability of finding Stentor at the locations where duckweed grows. 2. Collect at least 2 L of water in a container that is easy to pour from. 3. After detritus and particulate matter have settled to the bottom of the container, gently fill a few smaller containers to examine for Stentor, waiting for a few seconds after each collection for the detritus to settle in the large container. 4. Once the collection of samples is completed at a location, move to a new location that is at least 10 m away and repeat the collection of samples there. Note: Not every pond has a Stentor population, so multiple ponds may have to be sampled. 5. Return with the samples to the lab and transfer the water from the containers into individual Petri dishes. 6. In each Petri dish, search for Stentor under a stereomicroscope with oblique light at 5X magnification. Transfer individual Stentor cells using a 1 mL pipette into a well of a glass spot plate containing at least 100 µL of commercially available pasteurized spring water (PSW). Note: Stentor cells have a trumpet-like shape when they are attached to a substrate (Figure 1). Swimming Stentor cells are less extended than the cells attached to a substrate (Video 1). 7. Wash Stentor in PSW at least 3x. Perform the washes by removing about 90% of the water from the well while keeping Stentor cells in the well, followed by adding 500 µL of PSW into the well. Note: Before handling Stentor using pipettes with pipette tips of 10 µL or smaller, cut about 0.5 mm off the end of the pipette tip with scissors to avoid wounding the cells due to shear forces that are generated as a large cell flows through too small of a tip opening.

Prepare
Chlamydomonas before each feeding of Stentor. 1. Transfer 1 mL of Chlamydomonas culture prepared as in Step 1.1 into a 1.5 mL microcentrifuge tube. Centrifuge at 2,095 x g for 3 min. 2. Remove the supernatant and resuspended the pellet in 1 mL of PSW. Centrifuge at 2,095 x g for 3 min. Remove the supernatant and resuspend the pellet in 500 µL of PSW. Note: Thus, washed and concentrated Chlamydomonas will be referred to as "prepared Chlamydomonas" in the following steps. TAP media is detrimental to Stentor, thus washing Chlamydomonas before feeding Stentor is important.
Remove as many Stentor as possible into a new culture dish. Then wash the cells three times.
Wash Stentor thoroughly when receiving them, even when purchasing them from a commercial supplier.
Culture is overwhelmed by fungus or other small organisms.
Identify a corner where there are the least Stentor. In that area, remove as much media as possible without removing many Stentor and add in new PSW. Mix gently but throughly to distrubute the invading organisms. Skip the next feeding and the Stentor will eat them.
Observe the culture regularly and remove small contaminants by exchanging media for fresh PSW.
Stentor are laying on top of each other. Split the culture so that the concentration is less than 20 cells/mL.
Split cultures more often.
Stentor cells are mating. Feed every 4 days instead of every 5 days.
Culture has a lot of dark waste material. Remove as much of the dark material, Stentor waste, as possible without removing the cells.
If Stentor are attached to the dark material, pipette up and down to release the Stentor cells from their waste and then remove the media with the waste without removing the cells. Or, remove those Stentor and feed accordingly less.
Feed Stentor 1 mL less food per 100 mL of culture.
Remove as much media as possible without removing many Stentor cells and add new PSW.
Exchange the media regularly.

Discussion
Culturing Stentor presents a number of challenges. First, to perform experiments that require large numbers of cells, one needs to maintain a large number of Stentor cultures, as cultures become unhealthy when Stentor concentration exceeds 20 cells/mL. Second, the organisms that can contaminate Stentor cultures often divide faster than Stentor and overwhelm the culture (a common contaminant is rotifers). Thus, it is necessary to inspect the culture under a microscope periodically and remove the contaminants. Occasionally, a new culture needs to be started from a small number of cells rescued manually from a contaminated culture. This increases the time required to maintain healthy Stentor cultures. Third, expanding a single cell into a 400 mL culture requires at least one month because Stentor cell cycle is 3 -5 days. Fourth, unlike other model ciliates, Stentor rarely go into cyst form and they cannot be frozen.
An important aspect of culturing Stentor is the selection of an appropriate food organism. Various methods for culturing Stentor were previously described. One of them suggests to use skim milk to culture bacteria which then feed Stentor. Such a technique is effective in culturing Stentor; however, application of genomic techniques requires pure samples to avoid confusion resulting from genomic reads from undefined food organisms. Using the current protocol, Stentor can be grown in mass and, because their food, Chlamydomonas, has been sequenced, the presence of genomic contamination from the food organism can be detected and controlled for. For unknown reasons, Tartar had to replenish his stocks by returning to where he found Stentor before. With the current protocol, we have been able to keep Stentor for years.
Regeneration experiments with Stentor are generally straightforward, but there are a few critical details to keep in mind. In regard to Section 2, cutting Stentor cells in half can be mastered in minutes. Mastering more advanced microsurgery procedures of Stentor may require a week of practice. While performing a sucrose or urea treatment (Section 3), if at the beginning of imaging most cells still have their membranellar bands, increase incubation time by 10 -30 s for when sucrose treatment is performed next. Do not increase the time of sucrose treatment beyond 3 min incubation because it will result in cell death.
Imaging of Stentor regeneration requires methods to image large cells over long periods of time. The imaging method detailed in Section 4 can only be used with an upright microscope. If an inverted microscope is available instead, then cells can be placed on a slide or coverslip in small chambers. One method is to create a chamber out of petroleum jelly and cover with another coverslip to prevent evaporation. Alternatively, a chamber for an individual cell can be made by making a hole with a hole punch of the desired size in a 1 x 1 cm 2 square of silicon spacer (Table of Materials), placing the spacer on a coverslip, putting cells in the chamber, and covering the chamber with another coverslip. When imaging, if Stentor are not in the correct orientation to observe the characteristic features of each stage of regeneration, tap the plate firmly to make the cell contract. Watch the cell extend to identify the stage of regeneration (full extension takes about 45 s). If the cell is still in the wrong orientation, repeat the tapping. The images shown in Figure 1 and Figure 6 were taken by a stereo zoom microscope; however, all experiments detailed can be performed using a 5X dissecting stereo microscope.
Another challenge besides culturing and imaging Stentor is the tracking of regeneration, specifically the amount of time necessary to identify stages. If the regenerating cell is perfectly oriented with the region of the oral primordium clearly visible, identification of the stage takes a few seconds. Sometimes, however, the Stentor cell is in an orientation preventing clear assignment of regeneration stage, thus taking more time to identify. The significant amount of time required to stage individual regenerating cells might delay the quantification of all regenerating cells, thus decreasing the temporal precision of staging and requiring the observer to wait and re-image the cell after it has moved to a new orientation. Consequently, this experiment is both time and labor intensive. For these reasons, it would be highly desirable to develop automated