Stentor coeruleus has long been employed as a model organism to understand unicellular regeneration. While the timeline for visual morphological regeneration is known, the timeline for functional regeneration remains unknown. This assay tracks the motility of center population undergoing synchronized regeneration such that the recovery of loss function can be quantified.
It can be adapted for use with other cell types. The lack of a secure seal of the imaging chamber will result in wells leaking into each other or air bubbles forming inside of the wells. For researchers unfamiliar with the use of silicone spacers we strongly recommend that PSW or other clean medium be pipetted into each of the wells to test for leakage before use.
The construction of the sample chambers is easier shown than verbally described. As is the process for extracting motility patterns from the acquired video data. To begin, cut a piece of 250 micrometer thick silicone spacer sheet slightly smaller in both height, and width than a microscope slide.
Use a whole punch to create circular wells leaving sufficient space between neighboring wells to ensure a good seal. Initiate regeneration of oral apparatus by incubating stentor coeruleus cells in 10%sucrose or 2%uria for two minutes. Then wash three times with fresh medium.
Gently pipette approximately 10 stentor into each well. Taking care to match the sample volume to the well volume as closely as possible. Close the wells by gently lowering a piece of cover glass over the wells starting from one edge.
Use a narrow and slightly flexible object, such as a 10 microliter pipette tip to press down on the cover glass where it contacts the silicone sheet to ensure a good seal. Place the sample on the microscope stage and set the microscope to the lowest available magnification so that one well fits in the frame. Begin the time lapse and acquire a ten second video at 30 frames per second of each well at each time point.
To calibrate, acquire a clear image of a calibration slide or a clear ruler at the same magnification settings as the videos. Using any image viewing program, count the number of pixels between marks of a known physical distance. Download the two scripts, track cells, and clean traces to an easy to remember location on the computer and transfer the data videos to this computer as well.
Organize the data videos into folders. One for each time point. Use the script track cells to perform automated cell tracking in the data videos by opening track cells and running the script Choose add to path"if prompted by a popup window which will typically only happen when the script is run for the first time from a given folder.
When prompted, select one or more data videos to initiate tracking. Navigate to the data videos and once satisfied with the list of files click on open"To manually reject anomalous or false traces, open clean traces in the MATLAB editor window by double clicking on the file. At the prompt select data folder output by track cells.
Navigate to one of the created folders and select it. When prompted, enter one to keep the trace and zero to reject the trace. Then press enter to move on to the next trace.
Begin by downloading the script titled spaghetti plots. Open and run the script. When the file browser window pops up with the prompt, select folder to open.
Navigate to the folder of one of the time points. When prompted, calibration, what is the number of pixels per millimeter? Type in the calibration value and press enter.
Adjust the axes of the plot as necessary by changing line 55 of the script, which by default is set to RR equals four. for including a radius of up to four millimeters. Save the plot.
The results show the collective motility of the stentor cell population at two, three, seven, and eight hours into the oral apparatus regeneration process. These plots show both the expected increase in the number of motile individuals and the fourfold increase in the range of the most motile cells within the population. The fastest swimming cells, if started close to the edge, are restricted from swimming entirely in a straight line and their movement range appears smaller.
The tract cells were classified into three distinct categories, non-motile with no visible hold fast, non-motile with visible hold fast, and motile. The result showed a stacked histogram summarizing relative cell counts across these categories as a function of ours into regeneration. At later times, a significant number of cells were found in colonies with visible hold fasts strongly suggesting they had explored the environment and preferentially attached near others of their kind.
It is important to select the sample well dimensions that work well for stentor coeruleus which are approximately one millimeter in size. This includes silicone spacer thickness 5/16th inches in diameter, and 10 cells per well. These numbers should be adjusted when adapting this protocol for other cell types.
This assay can be performed in conjunction with RNA-I to investigate and compare the regeneration of different stentor phenotypes.
