The aim of the following experiment is to analyze cellular viscoelastic properties under different physiological and pathological conditions. This is achieved by applying negative pressure to the cell membrane via aass micro pipette connected to a pressure transducer that has been constructed from glass capillaries using a micro pipette puller. The pipette is mounted onto a fine micro manipulator positioned on a stage of an inverted microscope, and the tip of the pipette is brought into close proximity of a cell.
Next, a seal is created between the tip of the pipette and the cell membrane membrane deformation into the pipette is then measured in response to an applied step of negative pressure. Results are obtained that show effects of different treatments on cellular biomechanical properties based on the degree of membrane deformation in response to well-defined force. The impact of our lab is to investigate the impact of dyslipidemia on biophysical and biomechanical properties of vascular endothelial cells, and we're going to tell you today about a method that is called micropipet aspiration that we use to measure cell stiffness.
The implications of this technique extend toward the basic understanding of cellular biomechanics in multiple mechanisms such as angiogenesis, mechanical sensing, and migration, and more. Though this method can provide insight into cellular biomechanics, it can also be applied to other systems such as model membranes, intracellular organelles, or tissues. To visualize the cell membrane and observe the membrane projection into the pipette, vascular endothelial cellular membranes are stained with a lipophilic fluorescent dye.
To begin dilute stock dye dye to a working concentration with a warmed PBS solution sonicate the mixture for five minutes to break down the D aggregates. Following a five minute spin in the micro centrifuge, remove the supinate wash vascular endothelial cells by adding 0.5 milliliters of PBS. After five minutes, aspirate the solution and proceed to wash twice more.
Then add the dice solution to the cells and incubate for 30 minutes and a 37 degrees Celsius incubator. Following incubation, wash the cells again with PBS as before. Micro pipettes are pulled by heating a glass capillary in the middle.
When the glass starts to melt, the two halves of the capillary are pulled apart to generate two micro pipettes. The tips of the pipettes used in these experiments typically range between two to six microns. External diameter, depending on the size of the cell, the the shape of the tip should approximate a cylindrical tube to begin inserter glass capillary into the pipette puller.
In this demonstration, the Suter P 97 horizontal pipette pull is used with an optimized program. This puller offers multiple programmable options to vary the velocity and other parameters of the pull. Proceed to activate the pull program.
After pulling each micro pipette, verify the shape of the tip under the microscope After fire polishing the tip, fill the micro pipette with a physiological saline solution such as PBS or non fluorescent growth media. Importantly, the solution should be supplemented with 30%serum that will allow the cell membrane to move smoothly into the pipette. In this demonstration, an inverted fluorescent microscope with 3D deconvolution capabilities is used with a video camera connected to a computer, a pressure transducer, a vibration free station, and a micro manipulator to begin mount cells into a micro aspiration chamber by lifting the cells from their substrates and pipetting them into a shallow longitudinal chamber that is mounted on the inverted fluorescent microscope.
The rationale to use a shallow longitudinal chamber is to allow a micro pipet to approach the cells at a very shallow angle as close to horizontal as possible. This is done to allow the membrane pulled into the micro pipette to be visualized on a single plane of focus. Place a filled micro pipette into a pipette holder connected to a power transducer by flexible tubing, with a diameter adjusted to the connector of the pipette holder for a tight fit.
At the beginning of each experiment, the pressure in the pipette is equilibrated to the atmospheric pressure, mount the pipette onto a micro manipulator that allows fine control of the pipette movements in the micron range range. Position the pipette as a shallow angle to the bottom of the chamber and bring the tip of the pipette to the center of the visual field. The shank of the pipette, which is a cylindrical part of the pipette tip into which the membrane is aspirated, should be aligned horizontally to the focal plane.
To accomplish this first position, the pipette at the shallowest angle possible and then flex the shank of the pipette against the bottom of the chamber. Because the shank is very thin, it is flexible enough to slide on the bottom of the chamber while approaching a cell. Slowly bring down the micro pipette to the side of a single cell using the course manipulator until near the plane of focus for the cell.
Then using the fine manipulator, move the micro pipette to the edge of the cell until the tip of the pipette gently touches the membrane. Take one image to observe the position of the pipette. Good seals are created when the whole tip of the pipette is in full contact with the cell surface and the contact is stable.
Next, apply a step of negative pressure using the transducer and maintain it until the membrane projection is stabilized. The amount of pressure needed to aspirate the membrane into the pipette varies depending on cell type and specific experimental conditions. Initial deformation is typically observed when applying pressure in the range of minus two to minus 15 millimeters of mercury.
When the pressure is applied, the membrane is gradually deformed into the pipette until it is stabilized at a certain length. A process that typically takes two to three minutes during this time, images of membrane deformation are required every 30 seconds to track the progression of the membrane that is pulled into the pipette. Finally, increase the pressure to the next level in two to five millimeters of mercury steps.
Repeat the whole procedure until the membrane projection detaches from the cell and moves into the pipette, at which point the experiment is stopped. To quantify the degree of membrane deformation, the aspirated length is measured from the tip of the pipette to the vertex of the circumference of the membrane projection. A larger pipette will apply more force on the cell membrane at the same level of pressure to account for the variability between diameters of the pipettes.
The aspirated length is normalized for the pipette diameter measured for each experiment to validate the use of the micro aspiration technique for substrate attached cells, it was tested with a disruption of F actin results in a decrease in cell stiffness of bovine aortic endothelial cells as estimated by this approach. Here, a typical series of fluorescent images of an endothelial membrane undergoing progressive deformation in response to negative pressure applied through a micro pipette is shown as expected. The membrane is gradually aspirated into the pipette and the aspirated length increases as a function of applied pressure time courses of the deformation show that disruption of f actin significantly increases the aspirated lengths of the projections under all pressure conditions.
Using this approach, it was discovered that cell stiffness increases when cellular membranes are depleted of cholesterol, whereas cholesterol enrichment had no effect.Here. A cholesterol enriched cell, a control cell and a cholesterol depleted cell are shown after reaching maximal aspiration lengths of minus 15 millimeters of mercury. The projections typically started to develop at minus 10 millimeters of mercury, and the time causes of membrane deformation could be measured for the negative pressures of 10, 15 and 20 millimeters of mercury.
Here, the maximal aspirated lengths are plotted as a function of the applied pressure. The maximal normalized length in depleted cells was significantly lower than that of control cells for pressures of minus 15 millimeters of mercury and minus 20 millimeters of mercury. Application of negative pressures above 25 millimeters of mercury resulted in detachment of the aspirated projection forming a separate sle.
The pressure level that resulted in membrane detachment was similar under different cholesterol conditions and unexpected observation. As previous studies show increase in membrane cholesterol enhances the stiffness of membrane lipid bilayers Once mastered, this technique can be done in two to three hours if performed properly. While attempting this technique, it's important to remember that this experiment is done on individual cells.
Therefore, three to five cells should be analyzed per condition, per experiment for a total of three total experiments.
