Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy
Published: July 31, 2023
Abstract
Source: Ahn, W. et al., Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy. J. Vis. Exp. (2021)
This video demonstrates sample preparation, fixation, and imaging of a macrophage cell that displays membrane ruffles using scanning electron microscopy, SEM. Detailed information about membrane morphology and organization is crucial for understanding the physiological condition of the cells.
Protocol
1. Cell line and cell culture 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. When cells become 80% confluent, wash the plate two times using sterile PBS. Detach cells by adding 1,000 µL of 0.5% trypsin-EDTA and in…
Disclosures
The authors have nothing to disclose.
Materials
| 0.5% Trypsin-EDTA | Gibco | 15400-054 | |
| 2% Glutaraldehyde | Electron Microscopy Sciences | 16320 | |
| 4% Paraformaldehyde | Santa Cruz Biotechnology | 281692 | |
| 5-(N-ethyl-N-isopropyl)-Amiloride | Sigma Life Science | A3085 | |
| Carbon Adhesive Tabs | Electron Microscopy Sciences | 77825-09 | |
| Dimethyl Sulfoxide | Corning | 25-950-CQC | |
| Dulbecco's Modified Eagle Medium | Cytiva Life Sciences | SH30022.01 | |
| Falcon 24-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate | Falcon | 353047 | |
| Fetal Bovine Serum | Gemini Bio | 900-108 | |
| HERAcell 150i CO2 incubator | Thermo Fisher Scientific | 51026282 | |
| Hummer Model 6.2 Sputter Coater | Anatech USA | 58565 | |
| JSM-IT500HR scanning electron microscope | |||
| Microscope Cover Glass | Thermo Fisher Scientific | 12-545-82 | |
| Pen Strep | Gibco | 15140-122 | |
| Phorbol 12-myristate 13-acetate | Millipore Sigma | 524400 | |
| RAW 264.7 macrophage | ATCC | ATCC TIB-71 | |
| Recombinant Human M-CSF | Peprotech | 300-25 | |
| Samdri-790 Critical Point Dryer | Tousimis Research Corporation | 8778B | |
| SEM Aluminum Specimen Mounts | Electron Microscopy Sciences | 75220 | |
| Sodium Cacodylate | Electron Microscopy Sciences | 12300 | |
| Trypan Blue Solution | Thermo Fisher Scientific | 15250061 |
Tags
Cite This Article
Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy. J. Vis. Exp. (Pending Publication), e21463, doi: (2023).