The overall goal of this procedure is to understand the molecular mechanisms by which low intensity pulsed ultrasound or lipo influences cell migration and thereby accelerates tissue repair. This is accomplished by first treating cells with cyclo heide for two hours to prevent de novo protein expression and thus auto stimulation of cells. The second step of the procedure is to spread detach cells for two hours on surfaces that have been coated with a carefully defined matrix ligand.
The third step is to stimulate the cells with ultrasound by gel coupling the cell culture plates to the ultrasound emitters. Results can show changes in focal adhesion formation due to ultrasound stimulation via staining cells for cullin and actin and temporal changes in rack one activation through active RAC one, pull down assay and subsequent western blossoming. This method can help us answer key questions in the adhesion signaling field, such as can physical manipulation of cells substitute for chemical stimuli such as growth factors or fibrin actin?
And how does that physical manipulation initiate cell migration? The implications of this technique extend towards therapy of chronic wounds because by understanding how ultrasound may affect cell behavior, we may be able to refine treatments to accelerate wound closure in individuals with wound healing defects such as elderly or diabetic patients. For biochemical assays directly coat the wells of plastic 3.5 centimeter plates with integrin ligand.
For immunofluorescence boiled glass cover slips three times in milli Q water. Then position four cover slips in each 3.5 centimeter plate derivatize the glass cover slips to ensure efficient ligand coating by adding two milliliters A one millimolar sulfa MBS to each, well allow the reaction to proceed for 30 minutes at room temperature, then wash the derivatized surfaces three times with PBS. Now coat the derivatized glass with two milliliters of integrin ligand.
Add the ligand to each well and incubate the plates overnight at four degrees Celsius the next day, prepare mono particulate BSA by heating 1%BSA, dissolved in PBS to 85 degrees Celsius for 13 minutes. Allow to cool and then pass through a 0.45 micron filter. First, wash the ligand coated surfaces three times with PBS and second block the surfaces with the prepared mono particulate BSA for 30 minutes.
To control the matrix environment throughout the experiment, protein synthesis should be blocked by an addition of 25 micrograms per milliliter cyclo heide to 80%confluent fibroblasts for two hours. Rinse the cyclo heide treated cells with PBS, trise the cells and pellet the cells. Re suspend the cells in A-D-M-E-M solution at a final concentration of 10, 000 cells per milliliter with the aid of a hemo cytometer.
Now incubate the resuspended cells at 37 degrees Celsius for 20 minutes. Then rinse the plates three times with PBS before seeding two milliliters of the cell suspension into each. Well allow cells to spread for two hours at 37 degrees Celsius, 5%carbon dioxide begin by warming the ultrasound emitter array and coupling gel.
Once warmed, apply a pea-sized blob of coupling gel to each emitter test the each emitter is functional. Using an ultrasound indicator diode detector, allow the gel to couple the emitter to the detector. If the emitter is functional, the DDE will light up.
The device has fixed settings and will deliver ultrasound at 1.5 megahertz pulsed at one kilohertz, delivering an intensity of 30 milliwatts per centimeter square. The device will automatically deactivate after 20 minutes. Place the wells on the emitter array, return array to an incubator, and allow temperature to equilibrate for 10 minutes switch on the ultrasound signal at the power supply.
The ultrasound signal will deactivate after 20 minutes. Mimicking the therapeutic regime if shorter stimulation times are required. Plates can be removed from the array at the appropriate time.
Points for longer stimulation times plates should be left on the array and allowed to develop. The emitter could be restarted, but we have seen no additional benefit when we have done this. In all cases, unstimulated plates should be used as a negative control.
For analysis of focal adhesion formation, apply a 20 minute ultrasound signal to S coated cover slips, and then incubate them for an additional 40 minutes at 37 degrees Celsius. This allows the focal adhesions to develop. Next, aspirate away the media.
Apply two milliliters of fixative and wait 14 minutes. Remove the fixative by aspiration and carefully rinse the cells three times with PBS. Apply the PBS along the edge of the well and swell the solution gently.
Once rinsed clean, transfer each cover slip from the 3.5 centimeter well to a 24 well plate for subsequent immunofluorescent staining. After the desired stimulation period, aspirate away the media and rinse the cells with PBS on ice lysa cells in the appropriate lysis buffer for your preferred biochemical assay. We typically analyze one activation by effector pull down assay.
Particularly this method could be used for biochemical analysis of any signaling pathway. In this protocol, we describe the induction of vinky and stained focal adhesions and RAC one activity by ultrasound for the focal adhesion experiments. The baseline position is that fibroblasts spread on a ligand of alpha five beta one integrin do not form finkel and containing adhesions unless a second fibroin connecting receptor syndicate four is engaged by addition of soluble ligand.
However, stimulation with ultrasound induces focal adhesion formation to the same extent as engagement of syncom four indicating that ultrasound stimulation can substitute for certain components of the fibrin neck independence signaling pathway. As a development of the basic assay, we show the same experiment repeated with fibroblasts lacking INDICUM four. These fibroblasts still respond to ultrasound but fail to respond to INDICUM four ligand.
This demonstrates that ultrasound can indeed bypass the need for certain fiber andum receptors. For the biochemical experiment, we demonstrate that ultrasound can induce activation of RAC one using a pull down assay precipitation of active RAC 1 0 10, 30, and 60 minutes after initiation of ultrasound stimulation reveals that RAC one activity peaks at 10 to 30 minutes before returning to the baseline. Bloss crude lysates for cullin ensures equivalent loading between time points.
The result resembles activation of RAC one by engagement of syndicate four, but is slightly more protracted than activation by fibronectin. Therefore, eight to 10 repeats are typically required to achieve significant data. While attempting this procedure, it's important to remember to eliminate additional stu stimuli by completely blocking protein synthesis with cyclo heide, avoiding contaminating culture media in the experiment and avoiding overcrowding of cells that result in cell cell contacts Following this procedure.
Other methods like scratch wound assays can be used to ask additional questions such as, how does ultrasound initiate cell migration?
