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…

Divulgazioni

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

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Citazione di questo articolo
Quantification of Membrane Ruffle Formation Using Scanning Electron Microscopy. J. Vis. Exp. (Pending Publication), e21463, doi: (2023).

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