The protocol described in this video will define the experimental procedures required for the life-size imaging of spreading cells and the use of a computational tool that quantifies cells spreading dynamics in an unbiased and automated fashion. The cell spreading asset allows for the continuous tracking of cell edge movement and morphological changes during cell spreading, which is a feature that is missing in most existing cell spreading assays. As well, this protocol implements the use of automated computer processing and analysis, providing information on cell circularity, area and protrusion retraction cycles of spreading cells.
Such automated processing reduces bias in data analysis and provides a robust method of analyzing cell spreading dynamics for a large number of cells. When combined with drug treatments or gene science and techniques, this protocol is amenable to large-scale speeds of molecular players regulating cell protrusions. For the given protocol, The cells used are mouse embryonic fibroblasts that genetically encode PH-Akt-GFP, which allows for the fluorescent tagging of the plasma membrane.
Prior to the beginning of the cell spreading assay, culture a dish of cells to 90%confluency. Once the cells have attained the proper confluency, flame a 22 by 22 millimeter cover slip and place it into a 35 millimeter cell culture dish. Coat the cover slip with fibronectin that has been diluted in PBS to a concentration of 2.5 micrograms per milliliter.
Place the dish with the cover slip into a 37 degree incubator for one hour. After one hour aspirate the fibronectin making sure not to touch the cover slip with the pipette tip. Wash the dish with PBS by gently pipetting around the cover slip two to three times.
For cell seeding, begin by aspirating the cell culture media from the dish of cells. Afterwards, wash the dish with warm PBS. Add 650 microliters of trypsin EDTA to the dish and tilt the dish to evenly distribute the enzyme.
Place the dish with the trypsin into the incubator for one minute. After incubation you will first need to add 10 milliliters of cell culture media into a centrifuge tube. Next, quickly add another 10 milliliters of media into the dish in order to quench the trypsin.
To dilute the cells that will be seeded onto the cover slip, pipette one milliliter of the dish's contents into the centrifuge tube. From the tube, pipette approximately 500 to 1, 000 microliters of diluted cells into the dish with the cover slip. Ensure that the cover slip is at a 10%confluency or 50, 000 cells per milliliter and adjust the volume of diluted cells as needed.
The purpose of these cells is to establish a plane of focus during image acquisition. With the remaining cells in the dish, passage one fifth of the cells into one small dish per treatment. These will be the cells that will be analyzed for spreading dynamics.
Place the passage dishes and the cover slip dish into an incubator for eight to 24 hours. To test the importance of Arp2/3 for cell spreading, first add five milliliters of cell culture media into a control and a treatment centrifuge tube. Next add 20 milliliters of dmem that is lacking phenol red into each of two larger centrifuge tubes.
Pipette either the drug CK-666 which is an inhibitor of the Arp2/3 complex or the controlled treatment such as DMSO into the small and large tubes. To begin pharmacological treatment of the cells, remove the passage dishes that were incubating overnight. Aspirate the cell culture media from all of the dishes and wash the dishes with warm PBS.
Take the small tubes containing either CK-666 or control supplemented media and add the contents of the relevant tube into each of the passage dishes. Label each of the dishes with the correct drug treatment and then place the dishes back into the incubator for an additional hour After one hour incubation, aspirate the drug supplemented media from each of the dishes. Next, add warm PBS to all of the dishes in order to thoroughly remove all of the remaining phenol red media.
Add 230 microliters of trypsin EDTA and place the dishes into an incubator for one minute. Remove the trypsinized dishes from the incubator and proceed to add five milliliters of the drug supplemented dmem that is lacking phenol red into a tube designated as tube B.Then add another five milliliters of the same media into the relevant dish to quench the trypsin. Pipette the media up and down several times in order to detach all of the cells from the dish.
Transfer all of the contents of the dish into another tube that has already been designated as tube A.In order to prepare a cell dilution that is appropriate for imaging, transfer one milliliter of cells from tube A into tube B.Repeat all of the dilution steps for each treatment. Place all of the tubes into the incubator for 45 minutes to allow cells to recover from trypsinization. Ensure that all parts of a cell magnetic chamber that can accommodate a 22 by 22 millimeter cover slip have been thoroughly cleaned before use.
Remove the dish with the cover slip from the incubator. Aspirate the cell culture media and wash the cover slip with warm PBS. Remove the cover slip using a pair of forceps and gently lay the cover slip onto the bottom plate of the magnetic chamber.
Next pick up the silicone gasket and place it on top of the cover slip. Attach the main body of the magnetic chamber onto the bottom plate. Add one milliliter of the dmem lacking phenol red corresponding to the relevant treatment to the magnetic chamber.
Take a lint-free tissue and carefully dab the enclosure between the main body and the bottom plate in order to check for any leaks. To complete the magnetic chamber, lower the transparent cover onto the main body. Lastly wipe the bottom of the cover slip with a tissue sprayed with water and another sprayed with 70%ethanol.
Preheat the stage top incubator of a confocal microscope to 37 degrees. Place the magnetic chamber on top of the objective. Prior to acquiring spreading dynamics, set focus to the already polarized cells in the green fluorescent channel.
This ensures that the spreading cells will be in focus once they attach to the cover slip. Remove the transparent cover of the magnetic chamber and pipette 500 microliters from tube B that was removed from the incubator. Place the transparent cover back on top.
To identify cells ideal for cell spreading analysis search for green halos which represent cells that have yet to attach to the cover slip or are in the earliest stages of attachment. Acquire images and save the files. After having completed the image acquisition of cell spreading, begin by running a compatible Python IDE such as Spyder.
For computational analysis of cell spreading dynamics, locate and open the cell spreading GUI file provided in the relevant script packages. Press the run button which will open the analysis GUI panel in the background. The GUI houses all of the settings needed for analysis.
To analyze cell area and circularity, input the acquisition file and the necessary settings in the cell spread area tab. Adjustments will have to be made depending on cell type and image acquisition parameters. After pressing run, the script will create a cell circularity and area versus time plot for all spreading cells.
For kymograph data, press the kymograph generator and analysis tab. This analysis requires input files to be cropped single cell image stacks. After inserting all of the settings and pressing run, the script will create multiple kymographs for the given cell.
On the left are representative cells from the DMSO and CK-666 treatments, automatically segmented using the provided script. As opposed to the isotropic and highly circular morphology demonstrated by control cells, Arp2/3 inhibited cells exhibit decreased circularity. Additionally, the automatically generated kymographs provide temporal resolution that is critical to understanding the dynamics of protrusions.
Control cells protrude persistently with little to no retractions. In contrast, Arp2/3 inhibition disrupts the stability of protrusions as indicated by the increase in retraction events throughout spreading. This videos should provide you with the understanding of how to properly seed cells, perform pharmacological treatments and prepare imaging and computational tools necessary for the quantification of cell spreading dynamics.
This protocol can be further combined with cytoskeleton fluorescent imaging and migration assays to identify the molecular players governing cell protrusions. Thank you for watching and good luck with your experiments.
