The overall goal of this procedure is to perme Alize a adherent cells. This is accomplished by first culturing experimental cells on cover slips in six well plates. The second step is to rinse the cells with one XPBS and cover them with the solution containing the biomolecule to be transferred into the cell.
Next, the inert gas jet is run over. The sample permeable the cells. The final step is to fix and stain the cells if desired.
Ultimately, immunofluorescence microscopy is used to show permeation efficiency and cell viability. The main advantage of this technique over existing methods such as proliferation or gene guns, as that this technique is easily set up and allows for the spatially restrictive permeation of adherent cells. In a culture, generally, individuals new to this technique may struggle because it could take time to perfect certain parameters such as the gas flow rate or capillary diameter to allow for efficient permeation in their particular applications.
The mass flow controller used in this procedure requires an external 12 vol power supply and is attached to a helium gas line to ensure a precise gas flow rate interfacing between the mass flow controller and the computer is performed by a data acquisition device. This unit is controlled by an input voltage ranging between zero and five volts. A control loop in the lab view program determines the required voltage at any given time.
Two, positioning linear slides are used to control the movement of the six well plate one for each direction. Each assembly consists of an MA 15 series Velm X uni slide assembly coupled with a stepping motor, and both assemblies are controlled by a single VELM XVXM stepping motor controller. The controller allows for manual jogging of each motor or accepts a string of external serial commands.
The combination of which allows for specific movement patterns. Several patterns can be programmed into the lab view program requiring the user to provide only the desired torch speed as well as the path dimensions. Begin preparation of the equipment by opening both the helium cylinder and regulator turn on the motor controller.
Open the lab view program and verify all conditions. Next, make sure the platform is centered to check. Look at each motor stage and verify that each carriage is not near either stage end and then have the program center, the carriages.
If this is the first of the day, it is recommended to run the program once without cells to ensure the setup is working correctly. To prepare the gas Jett capillary nozzle height, remove the capillary from the holder and zero the height dial. Place the six well plate on the platform.
Replace the gas jet capillary and lower it to the bottom of the well until it hits the cover slip. Secure the capillary in the holder using the height dial. Raise the capillary three millimeters, mark this height on the capillary base.
Lastly, raise the capillary to a safe height and then remove the six well plate. The adherent cell line for this protocol should be cultured to confluence on glass cover slips in a six well plate using standard culturing techniques for helo cells. Seat six, well plates with two times 10 to the fifth cells per well and incubate for 48 hours before treatment.
On the day of treatment, prepare an appropriate volume of solution containing the desired vector. For dextrin studies, 80 micrograms per milliliter of 10 kilodalton green fluorescent dextrin is recommended, hence fourth. This solution will be referred to as vector solution.
Remove cell culture media from a well and rinse three times with one XPBS pipette 1, 370 microliters of vector solution into the well. This should result in a liquid depth of approximately 1.3 millimeters. Place the well containing the vector solution in the center of the platform.
Lower the gas jet nozzle to the previously marked level indicating a nozzle height of three millimeters above the slide. Set the helium flow rate in the lab view program and select the desired movement pattern to be used. Select run.
When ready to begin the permeation protocol, there will be an initial lag period during which the mass flow controller is preparing to provide the necessary flow rate For the success of this protocol, it's important to optimize flow conditions to minimize cell depth and cell detachment. Once the appropriate flow rate has been reached, the lab view program will activate the stepper motors to move the well in the desired permeation pattern. Once the platform has stopped, remove the six well plate from the platform.
Wait approximately 30 seconds and then remove the vector solution. Wash the well three times with PBS. After the third wash, fill the well with fresh culture media.
Repeat the permeation protocol for the desired number of experiments. Be sure to include control samples that consist of wells being filled with the vector solution without the gas jett being run over them. Leave the solution in the control wells approximately one minute before removing the solution.
Washing the cells with PBS and filling the well with fresh culture media. Return the six well plate to the incubator in the case of dextrin permeation incubate for 15 minutes. Subsequently, image cells with the appropriate microscope to investigate permeable efficacy.
Shown here are results of permeable of helo cells with 10 kilodalton green fluorescent dextrin using a helium gas jet with a 0.86 millimeter inner diameter. Capillary panels A, B, and C show results from running the helium gas Jett at three different outlet pressures and panel D is a control with dextrin added, but without exposure to the helium gas jet cells were counters stained immediately after permeable to visualize cell death. In these images, dextrin fluoresces green and dead cells fluoresce red.
It was observed in panels A and B that the permeation track width and efficiency increased with a higher dynamic pressure with only a slight increase in cell death. However, once a high dynamic pressure was reached as observed in panel c, heela cells were stripped from the cover slips and very little peripheral permeation occurred through scanning electron microscopy studies. Micropores were observed in the cell membrane.
In this example, permeation of heela cells was performed using a helium gas Jett with a 0.86 millimeter inner diameter capillary at an outlet pressure of 300 Pascals scanning electron microscopy images at a magnification of 50, 000 x were acquired. Panel A shows a control sample exhibiting a smooth cell surface while panel B shows the surface of a perme cell exhibiting a pore with an 84 nanometer diameter Once mastered, this technique should take 15 to 30 minutes if performed properly. Following the procedure, stains can be done in order to assess additional factors such as cell viability.
