Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy

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…

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