Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy
Transkript
Begin by placing sterile glass coverslips in wells of a 24-well plate using autoclaved forceps. Then, seed RAW 264.7 macrophages onto the coverslips at a density of 106 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, pre-treat 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 1-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 coverslips twice with ice-cold PBS. Then, incubate the coverslips in a fixative for 30 minutes at room temperature, followed by overnight incubation at 4 degrees Celsius.
On the following day, without disturbing the cell monolayer, gently wash, and then, incubate the coverslips in 500 microliters of 0.1 molar sodium cacodylate for 15 minutes. Following two washes with 500 microliters of distilled water, wash the coverslips twice in 500 microliters of a graded ethanol series with a 10-minute incubation in each wash.
To perform critical point drying, place the coverslips 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 0 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 0 degrees Celsius. Re-press the Fill and Purge buttons to turn them off. Then, close the carbon dioxide tank. Re-press the Cool button 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 button and wait until the pressure decreases to 0 pounds per square inch. Turn off the Critical Point Dryer, and remove the coverslips.
Next, using Carbon Adhesive tabs, mount the coverslips 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 button and adjust the Voltage knob until the gauge reads 10 milliamperes. Then, remove the coated coverslips from the chamber.
To visualize and quantify the membrane ruffles, insert the sample coverslips 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 3500x, and image the sample by clicking the Photo button.
