The micropatterning bast cell chirality assay is a powerful tool for studying multicellular chiral morphogenesis in development of disease. It is also important for cell research. This macropattern system is easy to fabricate and use, is capable of producing highly reliable and repeatable results, and it's also compatible with automated high-throughput processing for large sample size.
Start by cutting the titanium gold-coated slide into small square pieces of approximately 12 by 12 millimeters. Using a glass cutter, slide across the surface to leave a dented track, then hold the glass slide at both sides and bend at the dent to break into small quarters. To clean the slide, soak it with the gold side up in 100%ethanol contained in a Petri dish on an orbital shaker for at least 10 minutes.
After aspirating the ethanol, dry the slide by blowing with a nitrogen stream. Clean the polydimethylsiloxane or PDMS stamps with soapy water before drying the stamp with nitrogen gas. Dissolve 5.74 milligrams of octadecanethiol or C18 in 10 milliliters of 100%ethanol to make two millimolar C18 solution, then clean the PDMS stamps with 100%ethanol.
Soak the PDMS stamp with the pattern surface facing down in C18 solution for 10 seconds and then gently dry it with nitrogen gas for 60 seconds. Once dried, lay the stamp facing down onto the gold slide for 60 seconds before removal. To ensure proper stamping, gently tap the tweezers onto the stamp to properly transfer the patterns.
Next, prepare the humidity chambers by pipetting one milliliter of 70%ethanol into an inverted Petri dish lid. And using the tweezers, lay down a piece of parafilm to cover the surface ensuring no bubbles remain. Remove the excess ethanol if required.
Add 40 microliter droplets of EG3 solution for each quarter glass slide onto the parafilm in the humidity chamber, leaving enough space between droplets to account for the spacing of the gold slides. Use tweezers to place the C18 printed gold slide facing down onto the droplet, ensuring no bubbles remain under the slide. Gently push the slides together without lifting them to minimize evaporation.
After sealing the Petri dish tightly with parafilm, leave it at room temperature for at least three hours. In a biosafety cabinet, soak and rinse the gold slide facing up three times in 70%ethanol to remove EG3, then leave in ethanol for 10 minutes for sterilization. After aspirating the ethanol, add sterile PBS.
Prepare another humidity chamber with PBS instead of ethanol as demonstrated. Next, add 50 microliter droplets of fibronectin solution for each quarter slide onto the parafilm, leaving some space between droplets. Then place the slide facing down onto the droplet as demonstrated previously and leave the Petri dish in the biosafety cabinet for 30 minutes.
After rinsing the gold-coated slide thrice, place the slide facing up in PBS. Before seeding the cells, warm the media and trypsin in a water bath tempered at 37 degrees Celsius. For better cell attachment, soak the pattern slide in a 12-well plate containing culture media and warm it at 37 degrees Celsius in the incubator.
Once the cells are trypsinized, neutralize the cells with FBS containing media, pellet down the cells at 100 times G for three minutes and then resuspend the cell pellet in fresh media. Count the cells and dilute the cell suspension to achieve the concentration of 200, 000 cells per milliliter. Add 0.5 milliliters of the cell suspension to each well containing one gold slide.
Gently shake the plate a few times for uniform cell seeding before incubating for 15 minutes for cell attachment. After 15 minutes, check the cell attachment under a microscope, and if required, incubate for some more time. Once the cells are attached, aspirate out the media containing unattached cells from each well and add one milliliter of fresh culture media.
Culture the cells in the incubator for 24 hours and check confluency to determine if chirality has formed. When the required confluency is achieved, fix the cells by removing the culture media. After rinsing with PBS once, add 4%paraformaldehyde solution to the slide and incubate room temperature for 15 minutes before rinsing again with PBS three times.
To acquire the images, use a phase contrast microscope with camera functionality and capture each ring on the slide at high resolution. For chirality characterization, download the MATLAB code files, add the code folder and subfolders to the MATLAB path and open the ROI_selection. m file.
In line four, change the directory to the desired data folder. Change the image size in line 14 with the first two figures representing the inner circle size of the ring while the other two representing the outer. To determine the region of interest or ROI in the phase contrast images, execute the MATLAB code ROI_selection.
m by clicking the run button. Manually drag the selection square to fit the ring, then double click on the image to confirm the selection. After selecting ROI from all the images in the folder, a mat file will be generated to store the ROI information for each image.
Then open the analysis_batch. m file and change the directory of the folder as demonstrated before. Click on the run button to execute the code analysis_batch.
m to determine the chirality of multiple cellular ring patterns. A datatoexcel. txt file will be generated containing circular statistics for each ring, as well as the numbers of clockwise non-chiral and anti-clockwise rings.
The current findings demonstrate the utility of the developed ring pattern chirality assay experimentally as well as the sensitivity of this assay to alterations in the cytoskeleton. In the present study, it was found that by disrupting the actin polymerization with 50 nanomolar latrunculin A treatment, the mouse myoblast C2C12 cells exhibited an alteration of counterclockwise or CCW chiral bias into clockwise or CW.In addition, the human umbilical vascular endothelial cells or hUVECs that were treated with 12-o-tetradecanoyl-phorbol-13-acetate or TPA to activate the protein kinase C displayed a dose-dependent shift of cell chirality from CW to CCW. After cell seeding to the patterns, chemicals or drugs can be supplemented to the medium to study response, and the transwell system can be incorporated to study cell co-culture.
This assay provides an efficient tool for investigating multicellular chirality under different experimental settings offers important insights into the role of cell chirality in various biological phenomena and processes.
