Name: visualizing membrane ruffle formation using scanning electron microscopy
Uploaded: 2021-05-27T15:00:00
Description: Watch this Scientific Journal Video about Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy at JoVE.com

The authors thank Libby Perry (Augusta University) for her help with the SEM sample preparation. This work was supported by the National Institutes of Health [R01HL139562 (G.C.) and K99HL146954 (B.S.)] and American Heart Association [17POST33661254 (B.S.)].

NOTE: The following is a general protocol used to quantify membrane ruffle formation in RAW 264.7 macrophages using SEM microscopy. Optimization may be required for different cell types.
1. Cell line and cell culture
<ol>
	<li>Grow RAW 264.7 macrophages in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated Fetal Bovine Serum (FBS), 100 IU/mL of penicillin and 100 &#181;g/mL streptomycin in a humidified incubator at 37 &#176;C and 5% CO2. R...

<ol>
	<li>Bohdanowicz, M., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+phospholipids+in+endocytosis,+phagocytosis,+and+macropinocytosis.">Role of phospholipids in endocytosis, phagocytosis, and macropinocytosis.</a> Physiological Reviews. 93 (1), 69-106 (2013).</li><li>Canton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinocytosis:+New+Insights+Into+Its+Underappreciated+Role+in+Innate+Immune+Cell+Surveillance.">Macropinocytosis: New Insights Into Its Underappreciated Role in Innate Immune Cell Surveillance.</a> Frontiers in Immunology. 9, 2286 (2018).</li><li>Racoosin, E. L., Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-CSF-induced+macropinocytosis+increases+solute+endocytosis+but+not+receptor-mediated+endocytosis+in+mouse+macrophages.">M-CSF-induced macropinocytosis increases solute endocytosis but not receptor-mediated endocytosis in mouse macrophages.</a> Journal of Cell Science. 102, 867-880 (1992).</li><li>Yoshida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+signaling+during+macropinocytosis+in+response+to+M-CSF+and+PMA+in+macrophages.">Differential signaling during macropinocytosis in response to M-CSF and PMA in macrophages.</a> Frontiers in Physiology. 6, 8 (2015).</li><li>Ghoshal, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nox2-Mediated+PI3K+and+Cofilin+Activation+Confers+Alternate+Redox+Control+of+Macrophage+Pinocytosis.">Nox2-Mediated PI3K and Cofilin Activation Confers Alternate Redox Control of Macrophage Pinocytosis.</a> Antioxidant and Redox Signal. 26 (16), 902-916 (2017).</li><li>Bryant, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+induces+macropinocytosis+and+SNX1-modulated+recycling+of+E-cadherin.">EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin.</a> Journal of Cell Science. 120, 1818-1828 (2007).</li><li>West, M. A., Bretscher, M. S., Watts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+endocytotic+pathways+in+epidermal+growth+factor-stimulated+human+carcinoma+A431+cells.">Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells.</a> Journal of Cell Biology. 109 (6), 2731-2739 (1989).</li><li>Hagiwara, M., Nakase, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+induced+macropinocytosis+directs+branch+formation+of+lung+epithelial+cells.">Epidermal growth factor induced macropinocytosis directs branch formation of lung epithelial cells.</a> Biochemical and Biophysical Research Communications. 507 (1-4), 297-303 (2018).</li><li>Commisso, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinocytosis+of+protein+is+an+amino+acid+supply+route+in+Ras-transformed+cells.">Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells.</a> Nature. 497 (7451), 633-637 (2013).</li><li>Bosedasgupta, S., Pieters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+stimuli+reprogram+macrophage+phagocytosis+to+macropinocytosis+for+the+rapid+elimination+of+pathogens.">Inflammatory stimuli reprogram macrophage phagocytosis to macropinocytosis for the rapid elimination of pathogens.</a> PLoS Pathogens. 10 (1), 1003879 (2014).</li><li>Liu, Z., Roche, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinocytosis+in+phagocytes:+Regulation+of+MHC+class-II-restricted+antigen+presentation+in+dendritic+cells.">Macropinocytosis in phagocytes: Regulation of MHC class-II-restricted antigen presentation in dendritic cells.</a> Frontiers in Physiology. 6, 1 (2015).</li><li>Zeineddine, R., Yerbury, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+macropinocytosis+in+the+propagation+of+protein+aggregation+associated+with+neurodegenerative+diseases.">The role of macropinocytosis in the propagation of protein aggregation associated with neurodegenerative diseases.</a> Frontiers in Physiology. 6, 277 (2015).</li><li>Yerbury, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+aggregates+stimulate+macropinocytosis+facilitating+their+propagation.">Protein aggregates stimulate macropinocytosis facilitating their propagation.</a> Prion. 10 (2), 119-126 (2016).</li><li>Jayashankar, V., Edinger, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinocytosis+confers+resistance+to+therapies+targeting+cancer+anabolism.">Macropinocytosis confers resistance to therapies targeting cancer anabolism.</a> Nature Communication. 11 (1), 1121 (2020).</li><li>Kanlaya, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinocytosis+is+the+major+mechanism+for+endocytosis+of+calcium+oxalate+crystals+into+renal+tubular+cells.">Macropinocytosis is the major mechanism for endocytosis of calcium oxalate crystals into renal tubular cells.</a> Cell Biochemistry and Biophysics. 67 (3), 1171-1179 (2013).</li><li>Csanyi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD47+and+Nox1+mediate+dynamic+fluid-phase+macropinocytosis+of+native+LDL.">CD47 and Nox1 mediate dynamic fluid-phase macropinocytosis of native LDL.</a> Antioxidants and Redox Signaling. 26 (16), 886-901 (2017).</li><li>Francis, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruffles+induced+by+Salmonella+and+other+stimuli+direct+macropinocytosis+of+bacteria.">Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria.</a> Nature. 364 (6438), 639-642 (1993).</li><li>Mercer, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+entry+by+macropinocytosis.">Virus entry by macropinocytosis.</a> Nature Cell Biology. 11 (5), 510-520 (2009).</li><li>Liu, X., Ghosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+nanoparticle+delivery+by+oncogenic+KRAS-mediated+macropinocytosis.">Intracellular nanoparticle delivery by oncogenic KRAS-mediated macropinocytosis.</a> International Journal of Nanomedicine. 14, 6589-6600 (2019).</li><li>Desai, A. S., Hunter, M. R., Kapustin, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+macropinocytosis+for+intracellular+delivery+of+therapeutic+nucleic+acids+to+tumour+cells.">Using macropinocytosis for intracellular delivery of therapeutic nucleic acids to tumour cells.</a> Philosophical Transactions of Royal Society of London B: Biological Sciences. 374 (1765), 20180156 (2019).</li><li>Lin, H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+macropinocytosis+inhibitors+using+a+rational+screen+of+Food+and+Drug+Administration-approved+drugs.">Identification of novel macropinocytosis inhibitors using a rational screen of Food and Drug Administration-approved drugs.</a> British Journal of Pharmacology. 175 (18), 3640-3655 (2018).</li><li>Singla, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC&#948;-Mediated+Nox2+Activation+Promotes+Fluid-Phase+Pinocytosis+of+Antigens+by+Immature+Dendritic+Cells.">PKC&#948;-Mediated Nox2 Activation Promotes Fluid-Phase Pinocytosis of Antigens by Immature Dendritic Cells.</a> Frontiers in Immunology. 9, 537 (2018).</li><li>Ivanov, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inhibition+of+endocytic+pathways:+is+it+specific+enough+to+be+useful.">Pharmacological inhibition of endocytic pathways: is it specific enough to be useful.</a> Methods in Molecular Biology. 440, 15-33 (2008).</li><li>Araki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+3-methyladenine+on+the+fusion+process+of+macropinosomes+in+EGF-stimulated+A431+cells.">Effect of 3-methyladenine on the fusion process of macropinosomes in EGF-stimulated A431 cells.</a> Cell Structure and Function. 31 (2), 145-157 (2006).</li><li>Koivusalo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amiloride+inhibits+macropinocytosis+by+lowering+submembranous+pH+and+preventing+Rac1+and+Cdc42+signaling.">Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling.</a> Journal of Cell Biology. 188 (4), 547-563 (2010).</li><li>Fischer, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy.">Scanning electron microscopy.</a> Current Protocols in Microbiology. , (2012).</li><li>Yoshida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+ruffles+enhance+activation+of+Akt+by+growth+factors.">Dorsal ruffles enhance activation of Akt by growth factors.</a> Journal of Cell Science. 131 (22), (2018).</li><li>Nakase, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+macropinocytosis+induction+by+stimulation+of+epidermal+growth+factor+receptor+and+oncogenic+Ras+expression+potentiates+cellular+uptake+efficacy+of+exosomes.">Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.</a> Science Reports. 5, 10300 (2015).</li><li>Gold, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clathrin+independent+macropinocytosis-like+entry+mechanism+used+by+bluetongue+virus-1+during+infection+of+BHK+cells.">A clathrin independent macropinocytosis-like entry mechanism used by bluetongue virus-1 during infection of BHK cells.</a> PLoS One. 5 (6), 11360 (2010).</li><li>Mishra, R., Bhowmick, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Macropinocytosis+in+Prostate+Fibroblasts.">Visualization of Macropinocytosis in Prostate Fibroblasts.</a> Bio- Protocols. 9 (10), (2019).</li><li>Gordon, R. 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The present SEM imaging protocol provides a tool to visualize and quantify membrane ruffle formation, circular protrusions, and macropinocytic cups on the cell surface in vitro. Although the current protocol focuses on macrophages, studies have shown that membrane ruffle formation also occurs on various other cell types including dendritic cells, fibroblasts, neurons, and cancer cells11,12,14,<sup class='xref...

Macropinocytosis is an endocytic process responsible for internalizing a large amount of extracellular fluid and its content via the formation of dynamic and actin-driven plasma membrane protrusions called membrane ruffles1. Many of these membrane ruffles form cups that close and fuse back onto the cell and separate from the plasma membrane as large, heterogeneous intracellular endosomes also known as macropinosomes1. Although macropinocytosis is induced by growth factors such as macrophage colony-stimulating factor (M-CSF) and epidermal growth factor (EGF) in a wide range of cell types, an additional unique, calcium-dependent process known as constitutive macropinocytosis has been also observed in innate immune cells2,3,4,5,6,7,8.
The ability of cells to internalize extracellular material via macropinocytosis has been shown to play an important role in a variety of physiological processes ranging from nutrient uptake to pathogen capture and antigen presentation9,10,11. However, because this process is non-selective and inducible, it has also been implicated in a number of pathological conditions. Indeed, previous studies suggested that macropinocytosis plays an important role in Alzheimer&#39;s disease, Parkinson&#39;s disease, cancer, nephrolithiasis and atherosclerosis12,13,14,15,16. Moreover, certain bacteria and viruses have shown to utilize macropinocytosis to gain entry into host cells and induce infection17,18. Interestingly, stimulation of macropinocytosis can be also exploited for targeted delivery of therapeutic agents in various disease conditions19,20.
Previous studies have explored macropinocytosis by quantifying internalized fluorescently-tagged fluid-phase markers in the absence and presence of pharmacological agents that inhibit macropinocytosis using flow cytometry and confocal imaging21,22. Currently available pharmacological tools that inhibit macropinocytosis are limited to and comprise of 1) actin polymerization inhibitors (cytochalasin D and latrunculins), 2) PI3K blockers (LY-290042 and wortmannin) and 3) inhibitors of sodium hydrogen exchangers (NHE) (amiloride and EIPA)5,14,15,23,24,25. However, because these inhibitors have endocytosis independent effects, it is difficult to selectively determine the contribution of macropinocytosis to solute uptake and disease pathogenesis especially in vivo21.
Scanning electron microscopy (SEM) is a type of electron microscope that produces ultra-high-resolution images of cells using a focused beam of electrons26. In macropinocytosis research, SEM imaging is regarded as the gold standard technique to visualize topographical and morphological characteristics of the plasma membrane, quantify membrane ruffle formation, and investigate their progression towards macropinosome internalization. Furthermore, scanning electron microscopy combined with the quantification of solute uptake, in the presence and absence of macropinocytosis blockers, provides a reliable strategy to examine macropinocytotic solute internalization in vitro. This paper provides a detailed protocol on how to prepare cells for SEM, visualize the cell surface, quantify ruffle formation, and examine their progress towards cup closure and macropinosome internalization.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>2% Glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16320</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Santa Cruz Biotechnology</td>
			<td>281692</td>
			<td></td>
		</tr>
		<tr>
			<td>5-(N-ethyl-N-isopropyl)-Amiloride</td>
			<td>Sigma Life Science</td>
			<td>A3085</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Accuri C6 Flow Cytometer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Adhesive Tabs</td>
			<td>Electron Microscopy Sciences</td>
			<td>77825-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Corning</td>
			<td>25-950-CQC</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Cytiva Life Sciences</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate</td>
			<td>Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini Bio</td>
			<td>900-108</td>
			<td></td>
		</tr>
		<tr>
			<td>FitC-dextran</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1823</td>
			<td></td>
		</tr>
		<tr>
			<td>FM 4-64</td>
			<td>Thermo Fisher Scientific</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>HERAcell 150i CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>51026282</td>
			<td></td>
		</tr>
		<tr>
			<td>Hummer Model 6.2 Sputter Coater</td>
			<td>Anatech USA</td>
			<td>58565</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">JSM-IT500HR scanning electron microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-545-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>phorbol 12-myristate 13-acetate</td>
			<td>Millipore Sigma</td>
			<td>524400</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW 264.7 macrophage</td>
			<td>ATCC</td>
			<td>ATCC TIB-71</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Samdri-790 Critical Point Dryer</td>
			<td>Tousimis Research Corporation</td>
			<td>8778B</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM Aluminum Specimen Mounts</td>
			<td>Electron Microscopy Sciences</td>
			<td>75220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate</td>
			<td>Electron Microscopy Sciences</td>
			<td>12300</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas red-dextra</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1864</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 780 confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing membrane ruffle formation using scanning electron microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Here, we describe the results from the presented technique. Representative SEM images shown in Figure 1 demonstrate membrane ruffle formation in RAW 264.7 macrophages following treatment with PMA and M-CSF. Images were first captured at a magnification of 3,500x for quantification purposes and then at higher levels of magnification (8,500x to 16,000x) to show the plasma membrane at greater details. Pretreatment of macrophages with the macropinocytosis inhibitor EIPA attenuated membrane ruffl...

Watch this Scientific Journal Video about Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy at JoVE.com

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

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Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage ...

Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular ...