This method will enable researchers to quantify the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the extracellular matrix protein fibronectin during oxidative stress. This technique is versatile and can be adapted to examine cytoskeletal dynamics under a wide range of conditions. Furthermore, data acquisition and analysis are performed using common laboratory equipment and freely available software.
Understanding the mechanisms for how cells attach and spread on the ECM during alterations in redox status can provide valuable insight into normal and disease states, such as metastatic cancer. This method can be used to examine cell spreading and adhesion under different cell culture conditions, anchorage-dependent cell lines, and/oxidants to answer a broad range of biological questions. There are numerous reagents required to perform this technique.
Therefore, it is essential that all solutions are made in advance before beginning. In a BSL-II certified laminar flow hood set out a tissue culture-certified 24-well plate. Place one glass coverslip into each well.
Label the plate according to figure 1B of the text protocol. Next, pipette 500 microliters of the fibronectin solution into each well. Pipette the solution over each coverslip a few times to ensure an even coating and complete submersion.
Cover the plate with the lid. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for one hour. Then remove the plate from the incubator and aspirate the fibronectin solution from the wells.
Wash the wells three times using 500 microliters of 1X PBS per wash. Aspirate the final wash of 1X PBS, and block wells with 500 microliters of a 0.5%delipidated BSA solution at 37 degrees Celsius with 5%carbon dioxide for a minimum of 15 minutes. First, pre-warm the needed solutions in a water bath at 37 degrees Celsius.
In a BSL-II certified laminar flow hood, start with a confluent monolayer of REF52 cells in a 10-square centimeter dish and wash the cells twice with six milliliters of warm 1X PBS. Serum starve the cells for at least one hour in six milliliters of warm delipidated BSA solution. Next, wash the cells with six milliliters of warmed 1X PBS.
Aspirate the PBS and add 1.5 milliliters of warm 0.5%trypsin-EDTA solution. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for approximately two minutes. Observe the cells under a light microscope to ensure that the detachment is complete.
If the cells are still adherent after tapping the plate on the bench top, return them to the incubator for an additional two minutes. Pipette 1.5 milliliters of warm trypsin neutralizing solution, TNS, to the dish to stop trypsinization and collect detached cells. Pipette the solution up and down over the bottom of the plate numerous times to remove all of the remaining adherent cells.
If the cells appear clumpy, further triturate the cell suspension by gently pipetting up and down over the back of the dish. Next, count the cells using trypan blue exclusion in an automated cell counter. Remove an appropriate amount of cells to create a cell suspension with the cell density between 10, 000 and 30, 000 cells per milliliter in delipidated BSA in a 15-milliliter conical tube.
Use a fixed-angle rotor in a tabletop centrifuge to centrifuge the cells at 300 times g for five minutes. Aspirate the supernatant and re-suspend the cell pellet in seven milliliters of warm delipidated BSA solution. Then evenly divide the cell suspension into two 15-milliliter conical tubes, one for vehicle-alone control, and one for Ku55933.
Using a tube rotator, revolve the tubes for 90 to 120 minutes in a cell culture incubator at 37 degrees Celsius. 30 minutes before plating, add Ku55933 and DMSO to each tube to a final concentration of 10 micromolar. Return the cell suspension to the rotator for the remaining time.
Immediately prior to plating the cells, retrieve the plate from the incubator and aspirate the delipidated BSA solution. After this, remove 500 microliters of the cell suspension from each treatment group and add each one to one of the fibronectin-coated cover slips in the 24-well plate. Continue incubating the plate and the cell suspension at the previous conditions.
Then allow the cells to adhere to the coverslip for the desired length of time. After the desired time for adhesion has passed, aspirate the cell solution from each coverslip in the plate. Gently dispense 500 microliters of 3.7%paraformaldehyde solution onto each coverslip by pipetting down the sides of the wells, and wait 10 to 15 minutes.
Next, remove the paraformaldehyde solution and wash each coverslip twice with 1X PBS using 500 microliters of PBS per wash. Aspirate the PBS and permeabilized cells on each coverslip with 500 microliters of 0.2%Triton X-100 and 1X PBS for 10 to 15 minutes at room temperature. Then wash each coverslip three times with 1X PBS using 500 microliters of PBS per wash.
Block the cells on each coverslip with 500 microliters of immunofluorescence blocking buffer for 30 to 60 minutes. Dilute the primary anti-paxillin antibody in the blocking buffer at a ratio of one-to-250. Mix well and add 200 microliters of the antibody solution to each coverslip.
Incubate at room temperature for at least one hour. After this, aspirate the antibody solution and wash each coverslip with 500 microliters of 1X PBS for 10 minutes. Protect the samples from light from this point forward.
Dilute the phalloidin F-actin probe conjugated to the red fluorescent Alexa 594 dye and the goat anti-mouse 488 fluorescent secondary antibody in the same blocking buffer solution. Mix well and add 200 microliters of the antibody solution to each coverslip for 30 minutes. Aspirate the antibody solution and wash each coverslip with 500 microliters of 1X PBS for 10 minutes.
Aspirate the PBS and rinse the cover slips once with 500 microliters of deionized water. Then fix the cover slips onto microscope slides using anti-fade mounting medium containing DAPI. After fixation and staining with an anti-paxillin antibody, fluorescent 8-bit grayscale images of the REF52 cells are acquired.
Upon completion of all image processing steps, individual focal adhesions should be prominent, in focus, and readily distinguishable from one another. Grayscale fluorescence images of anti-paxillin and phalloidin F-actin probe stained cells are then taken after being plated on fibronectin. Prominent focal adhesions and stress fibers should be readily visible in REF52 cells after being allowed to adhere on fibronectin for 20 to 30 minutes.
F-actin enriched ruffles at the leading edge of cell membranes are indicated with an arrow. Similar fluorescent images of phalloidin F-actin probe and anti-paxillin staining are analyzed for the percentage of stress fibers in cell spreading. Notably, oxidant treatment causes a significant increase in stress fiber formation at all adhesion time points examined, and a decrease in cell spreading following 15 minutes of cell adhesion to fibronectin.
Plating at higher cell densities leads to cellular crowding which prohibits cells from fully spreading due to overconfluency. Notice that the cell edges are indistinguishable from adjacent cells. As a result, quantification of individual cells is precluded and spreading circumference cannot be accurately determined.
A separate cell line of mouse embryonic fibroblasts are held in suspension and then plated on fibronectin for 30 minutes. Cells were then fixed and stained with an anti-paxillin antibody. Note the out-of-focus cells.
Furthermore, the cross-reactivity of the anti-paxillin antibody with cellular debris will alter thresholding during quantitative image analysis and should not be included in the analysis. During image processing, use the look-up tool in Fiji to examine the image histogram while adjusting the brightness/contrast to avoid pitfalls related to signal saturation. Changes in adhesion and spreading may influence cell migration.
Methods to track single cell migration, wound healing, or chemotaxis invasion assays can determine if alterations in migration are occurring. Rho family GTPases regulate cell adhesion dynamics through their specific spatial temporal activation. Phenotypic changes in adhesion and spreading during oxidative stress may be suggestive of downstream regulatory roles for these proteins.
This method requires the use of BSL-II cell lines and hazardous chemical reagents. Researchers require chemical and biological safety training as determined by their institution's environmental health and safety office.
