The overall goal of this protocol is to transform non-fusogenic cover glass into a fusogenic surface that can be used to study macrophage fusion using any form of optical microscopy. This method can help to answer the fundamental questions related to the molecular mechanism of macrophage fusion. The main advantage of this procedure is that any microscope technique, particularly live imaging, can be used to study macrophage fusion.
Demonstrating the procedure will be James Faust, third fellow, and Arnat Balabiyev, a graduate student from my laboratory. To begin, in a well-ventilated chemical fume hood incubate high-stringency cover glass in 12-molar hydrochloric acid with sonication for one hour. Repeat the step two additional times with fresh 12-molar HCL.
Fill a separate beaker with ultrapure water, and add the cover glass. Then sonicate the cover glass for five to 10 minutes. Next, incubate the cover glass in pure acetone for 30 minutes with sonication.
Repeat the step two additional times. Then fill a separate beaker with sterile ultrapure water, and add the cover glass. Sonicate the cover glass for five to 10 minutes.
Incubate the cover glass in 100%ethyl alcohol for 30 minutes with sonication. Repeat this step two additional times. For longterm storage, place the acid-cleaned cover glass in a container willed with pure ethyl alcohol.
Alternatively, dry the cover glass with nitrogen gas, and store in a vacuum desiccator. Dissolve DMSO-free high-melting-point paraffin wax in ultrapure toluene. Using a glass pipette, apply enough paraffin working solution to a dry acid-cleaned cover glass to evenly coat the glass surface.
Decant the excess solution, and with nitrogen gas or air, dry the cover glass. With a lint-free wipe, polish the cover glass using three strokes along the X axis, then three strokes along the Y axis. Immediately before the experiment is conducted, use sterile ultrapure water to wash the cover glass, and subsequently sterilize it with ultraviolet light in a biological safety hood for 15 to 30 minutes.
Dry the acid-cleaned cover glass, and immobilize the glass on a flat surface. Using forceps, carefully immerse a gold transmission electron microscopy finder grid in a working solution of a hydrocarbon compound. Wick away excess solution by gently tapping the grid on filter paper, then immediately place the grid in the center of the cover glass.
Allow the toluene to dry for two minutes. Make sure the grid is bonded to the glass by gently inverting the glass. If the grid detaches, repeat the bonding using a new acid-washed cover glass.
After removing the TEM grid, examine the continuity of the micro pattern with low magnification. The micro pattern should appear continuous, but the material should not extend beyond the margins of the pattern. Next, use a plasma cleaner with vacuum-gas plasma to clean the cover glass according to the text protocol.
Then, using fine-tip forceps, carefully remove the grid from the glass surface to expose the micro pattern. With a step drill bit, drill a six to 10 millimeter circular hole with smooth edges in the bottom of a 35-millimeter plastic petri dish. Carefully mix and degas silicon elastomer according to the manufacturer's instructions.
Apply a thin coating of elastomer just proximate to the edge of the hole in the petri dish. The elastomer should appear as a continuous thin band surrounding the hole. Gently apply either a fusogenic or the dry acid-cleaned cover glass to the dish in order to cover the hole surrounded by elastomer.
Ensure that the cover glass appears flush with the bottom of the dish and extends beyond the diameter of the hole so that a substantial portion of the glass is in contact with the plastic. Cure the elastomer by baking it at 50 degrees Celsius for two to three hours. Inject eight-week-old C57 black-6 mice with 0.5 milliliters of a sterile solution of 4%Brewer's Thioglycolate as outlined in the text protocol.
72 hours later, after euthanizing the animal according to approved animal care and use guidelines, collect macrophage by interperitoneal lavage using ice-cold PBS supplemented with five-millimolar EDTA. Centrifuge the macrophages at 300 times G for three minutes, and resuspend the cell pellet in one milliliter of DMEM/F-12 supplemented with 15 millimolar heaps 10%FBS and 1%antibiotics. After counting and diluting the cells and applying them to the surfaces for 30 minutes, use HBSS 0.1%BSA to wash the surfaces, and replace the culture medium.
Then return the cultures to the incubator. Two hours later, aspirate the medium, and replace it with culture medium supplemented with 10 nanograms per milliliter of IL4 before imaging the cells. When cleaned as described in this video, the cover glass is exceptionally flat with no obvious surface features;whereas the absorption of long-chain hydrocarbons followed by surface polishing creates a degree of nanotopography seen here.
On clean cover-glass surfaces macrophages fuse at a very low rate. In contrast, after 24 hours in the presence of IL4, the macrophages applied to surfaces adsorbed with compounds containing long-chain hydrocarbons undergo robust fusion. Further, absorption of solvent alone does not make glass a fusogenic surface.
Here, full width at half maximum, or FWHM, of structures smaller than the diffraction limit of the light microscope is used to quantitatively assess the potential differences in resolution. Importantly, there is no apparent difference in FWHM of 100-nanometer fluorescent beads among the various surfaces, suggesting that the characteristics of glass required for fluorescence imaging were sufficiently preserved. In this experiment macrophages were applied to the surfaces containing long-chain hydrocarbons.
Then a form of super-resolution microscopy called 3D Direct Stochastic Optical Reconstruction Microscopy was carried out. These videos demonstrate that a variety of live-imaging techniques are feasible when macrophages are applied to the surfaces. Once mastered, modifying the glass to make the surface fusogenic can be done in half an hour if it is properly done.
While attempting this procedure it is important to remember to follow it carefully. Following this procedure, other methods, like fluorescence microscopy, can be performed in order to obtain views of fusion with molecule-specific contrast. This will enable experiments designed to identify proteins directly involved at the time of fusion.
After its development, this technique paved the way for investigators to acquire time-resolved views of macrophage fusion. In the future, this will help us to better understand the mechanism of macrophage fusion. After watching this video, you should be able to modify cover glass to study macrophage fusion.
Please don't forget that working with acid and toluene can be extremely hazardous, and PPE and proper ventilation should always be used while performing this procedure.
