Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy

1. Cell line and cell culture

1. Grow RAW 264.7 macrophages in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated Fetal Bovine Serum (FBS), 100 IU/mL of penicillin and 100 µg/mL streptomycin in a humidified incubator at 37 °C and 5% CO₂. Replace growth media every other day.
2. When cells become 80% confluent, wash the plate two times using sterile PBS.
3. Detach cells by adding 1,000 µL of 0.5% trypsin-EDTA and incubate the cell culture plate for 3-5 min in a humidified incubator at 37 °C.
4. Examine the plate under a light microscope to confirm cell dissociation. Add 10 mL of growth media containing 10% FBS to inactivate trypsin.
5. Collect the cell suspension in a 50 mL conical tube and centrifuge at 400 x g for 5 min at room temperature. Gently resuspend cells in the complete cell culture medium and determine the cell count and viability using Trypan Blue (0.4%) staining.
   NOTE: The recommended minimum cell viability for this experiment is >90%.
6. Place sterile glass coverslips in wells of a 24-well plate using autoclaved forceps. Seed cells onto coverslips at a density of 1 x 10⁶ cells/mL and incubate the plate overnight in a humidified incubator at 37 °C and 5% CO₂.
7. Change the media of each well with fresh 500 µL complete media before treatment. Pretreat macrophages with vehicle (DMSO or other solvents used to dissolve EIPA) or the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA, 25 µM) for 30 min.
8. Treat cells with vehicle and stimulators of macropinocytosis to promote membrane ruffling: phorbol 12-myristate 13-acetate (PMA, 1 µM, 30 min) and macrophage colony-stimulating factor (M-CSF, 100 ng/mL, 30 min).      
   NOTE: Alternative inhibitors [e.g., latrunculin A (1 µM, 30 min) or cytochalasin D (1 µM, 30 min)] and stimulators [e.g., epidermal growth factor (EGF, 100 ng/mL, 10 min) or platelet-derived growth factor (PDGF, 100 ng/mL, 15 min)] of macropinocytosis can be also used to characterize macropinocytosis in macrophages and other cell types. Cells may require overnight serum starvation prior to the treatment with physiological stimulators of macropinocytosis. It is important to note that the most effective concentrations and incubation times will have to be determined to stimulate macropinocytosis in other cell types.

2. SEM fixation

1. After the treatment, aspirate the media from wells and wash coverslips with ice-cold PBS twice.
2. Fix cells (4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate) for 30 min at room temperature, followed by overnight incubation in the fixative at 4 °C.
3. Gently wash and incubate coverslips with 500 µL of the following reagents without disturbing the cell monolayer.
   1. Wash once in 0.1 M sodium cacodylate and incubate for 15 min.
   2. Wash twice with dH₂O with 10 min incubation in each wash.
   3. Wash twice with 25% ethanol with 10 min incubation in each wash.
   4. Wash twice with 50% ethanol with 10 min incubation in each wash.
   5. Wash twice with 75% ethanol with 10 min incubation in each wash.
   6. Wash twice with 80% ethanol with 10 min incubation in each wash.
   7. Wash twice with 95% ethanol with 10 min incubation in each wash.
   8. Wash twice with 100% ethanol. Perform 10 min of incubation in each wash.
      NOTE: Changing/replacing of reagents should be done fast to prevent air drying of cells. Coverslips can be left in 100% ethanol for several days at 4 °C. For long-term storage, add additional 100% ethanol and cover wells with parafilm to prevent air drying of the sample.

3. Critical point drying

1. Place coverslips in a Critical Point Dryer and cover them with 100% ethanol. Press the Power button and open the CO₂ tank.
2. Press the Cool button for approximately 30 s until the temperature decreases to 0 °C. Press the Fill button until a bubble appears in the chamber window.
3. Press the Purge button until the smell of ethanol from the purge exhaust disappears. Press the Cool button again until the temperature decreases to 0 °C.
4. Repress the Fill and Purge buttons to turn them off and close the CO₂ tank. Repress the Cool button to turn it off and press the Heat button.
5. Set the temperature at 42 °C and pressure at 1,200 psi. Once the pressure and temperature stabilize, press the Bleed button to allow the pressure to decrease slowly.
6. Once the chamber pressure reaches 150 psi, press the Vent button and wait until the pressure decreases to 0 psi. Turn off the critical point dryer and remove the coverslips.
7. Mount coverslips on SEM aluminum specimen mounts using Carbon Adhesive tabs and subject to sputter coating using gold/palladium in a sputter coater.
8. Turn on the Power button and wait until the vacuum reaches 30 mTorr. Flush the chamber to remove humidity and air by turning off the gas switch and turning the Fine gas valve counterclockwise.
9. Once the vacuum increases to 200 mTorr turn off the gas switch and wait until the vacuum reaches 30 mTorr. Repeat this step 3 times.
10. Push the Timer button and adjust the Voltage knob until the gauge reads 10 mA. Remove coated coverslips from the chamber.     
    NOTE: Follow the manufacturer's instructions to perform critical point drying and sputter coating of the sample.

4. Imaging and quantification

1. Insert sample coverslips into the chamber of a scanning electron microscope. Close the door and press the Evac button.
2. Open the SEM operation software. Set the accelerating voltage to 15 kv and the working distance to 10 mm.
3. Press the Coordinates button and move around the controller until the cells appear in the center of the observation screen. Set the magnification to 3,500x and image the sample by clicking the Photo button.
   NOTE: Use a higher level of magnification (8,500x to 16,000x) to show the plasma membrane at greater detail.