The present SEM imaging protocol provides a tool to visualize and quantify membrane ruffle formation, circular protrusions, and macropinocytic cups on the cell surface in vitro. SEM provides high-resolution images of the cell surface that can be used to to visualize macropinocytic membrane activities. This technique can be used to discover new signaling pathways regulating macropinocytosis, and identify novel stimulators and inhibitors of micropinocytosis.
Begin by placing sterile glass cover slips in wells of a 24-well plate using autoclaved forceps. Then seed raw 264.7 macrophages onto the cover slips at a density of 10 to the sixth cells per milliliter and incubate the plate overnight in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. On the following day, replace the media in each well with 500 microliters of fresh complete media, then pretreat the macrophages for 30 minutes with a vehicle control such as DMSO or a macropinocytosis inhibitor such as EIPA.
Next, to promote membrane ruffling, treat the cells for 30 minutes with macropinocytosis stimulators, such as a one-micromolar PMA solution or 100 nanograms per milliliter of macrophage colony stimulating factor. To fix the cells for scanning electron microscopy, aspirate the media from the wells and wash the cover slips twice with ice-cold PBS, then incubate the cover slips in a fixative for 30 minutes at room temperature followed by overnight incubation at four degrees Celsius. On the following day, without disturbing the cell monolayer, gently wash and then incubate the cover slips in 500 microliters of 0.1-motor sodium cacodylate for 15 minutes.
Following two washes with 500 microliters of distilled water, wash the cover slips twice in 500 microliters of a graded ethanol series with a 10-minute incubation in each wash. To perform critical point drying, place the cover slips in a critical point dryer and cover them with 100%ethanol. Then, press the power button and open the carbon dioxide tank.
Press the cool button for approximately 30 seconds until the temperature decreases to zero degrees Celsius. Then, press the fill button until a bubble appears in the chamber window. Next, press the purge button until the smell of ethanol from the purge exhaust disappears.
Then, press the cool button again until the temperature decreases to zero degrees Celsius. Re-press the full and purge buttons to turn them off. Then, close the carbon dioxide tank.
Re-press the cool bottom to turn it off, then press the heat button. Set the temperature to 42 degrees Celsius and pressure to 1, 200 pounds per square inch. Once the pressure and temperature stabilize, press the bleed button to allow the pressure to decrease slowly.
Once the chamber pressure reaches 150 pounds per square inch, press the vent bottom and wait until the pressure decreases to zero pounds per square inch. Turn off the critical point dryer and remove the cover slips. Next, using carbon adhesive taps, mount the cover slips on the aluminum specimen mounts for scanning electron microscopy, then proceed to sputter coating using gold or palladium in a sputter coater.
Turn on the power button of the sputter coater. After the vacuum reaches 30 millitorrs, flush the chamber to remove humidity and air by turning off the gas switch and turning the fine gas valve counterclockwise. Once the vacuum increases to 200 millitorrs, turn off the gas switch and wait until the vacuum reaches 30 millitorrs.
Then, flush the chamber again to remove humidity and air as demonstrated. After flushing the chamber three times, push the timer bottom and adjust the voltage knob until the gauge reads 10 milliamperes. Then, remove the coated cover slips from the chamber.
To visualize and quantify the membrane ruffles, insert the sample cover slips into the chamber of a scanning electron microscope, close the door, and press the evac button. Open the microscope operating software and set the accelerating voltage to 15 kilovolts and the working distance to 10 millimeters. Press the Coordinates button and move around the controller until the cells appear in the center of the observation screen.
Set the magnification to 3, 500 X and image the sample by clicking the Photo button. Shown here are representative scanning electron microscopy images, demonstrating membrane ruffle formation in raw 264.7 macrophages following treatment with PMA and macrophage colony stimulating factor. Pretreatment of macrophages with the macropinocytosis inhibitor EIPA attenuates membrane ruffle formation.
During ruffle formation, the plasma membrane undergoes distinct morphological stages, including a sheet-like membrane protrusion, a C-shaped membrane ruffle, and a macropinocytic cup. Membrane ruffle formation following PMA and macrophage colony stimulating factor treatments can be quantified using scanning electron microscopy, whereas macropinosome formation can be confirmed by alternative imaging techniques, such as confocal microscopy using Texas red dextran and FM4-64. The blue arrows indicate membrane ruffling, while the yellow and green arrows point to the micropinosomes.
Finally, macropinocytosis can be confirmed and quantified through flow cytometry using a fluorescent fluid phase marker such as FITC or Texas red dextran. In addition to SEM, live cell imaging and flow cytometry analysis of fluorescently-labeled dextran internalization can also be performed to investigate and quantify macropinocytosis.
