The authors wish to thank the Biosecurity Tick Colony (Queensland Department of Agriculture &amp; Fisheries, Australia) for the provision of Rhipicephalus microplus ticks utilized for this study, and Lucas Karbanowicz for assistance with video filming.

1. Dissection of the Gut Epithelium from R. microplus

<ol>
	<li>Collect semi-engorged ticks from cattle on the day of experiment. Dissect ticks within 24 h after removal from the host.</li>
	<li>Adhere a strip of duct tape to the bottom of the 92 mm x 16 mm Petri dish. Add a drop of super glue to the tape. Place the tick, ventral side down on the super glue, allow to dry for 2 min.</li>
	<li>Pour 100 mL of phosphate buffered saline (PBS) into the petri dish, or until the tick is completely subm...

<ol>
	<li>Rodriguez-Valle, 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=Efficacy+of+Rhipicephalus+(Boophilus)+microplus+Bm86+against+Hyalomma+dromedarii+and+Amblyomma+cajennense+tick+infestations+in+camels+and+cattle.">Efficacy of Rhipicephalus (Boophilus) microplus Bm86 against Hyalomma dromedarii and Amblyomma cajennense tick infestations in camels and cattle.</a> Vaccine. 30, 3453-3458 (2012).</li><li>De Rose, 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=Bm86+antigen+induces+a+protective+immune-response+against+Boophilus+microplus+following+DNA+and+protein+vaccination+in+sheep.">Bm86 antigen induces a protective immune-response against Boophilus microplus following DNA and protein vaccination in sheep.</a> Vet. Immunol. Immunopathol. 71, 151-160 (1999).</li><li>Garc&#237;a-Garc&#237;a, J. 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=Sequence+variations+in+the+Boophilus+microplus+Bm86+locus+and+implications+for+immunoprotection+in+cattle+vaccinated+with+this+antigen.">Sequence variations in the Boophilus microplus Bm86 locus and implications for immunoprotection in cattle vaccinated with this antigen.</a> Exp. Appl. Acarol. 23, 883-895 (1999).</li><li>Abbas, R. Z., Zaman, M. A., Colwell, D. D., Gilleard, J., Iqbal, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acaricide+resistance+in+cattle+ticks+and+approaches+to+its+management:+The+state+of+play.">Acaricide resistance in cattle ticks and approaches to its management: The state of play.</a> Vet. Parasitol. 203, 6-20 (2014).</li><li>Kearney, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Acaricide (chemical) resistance in cattle ticks. , (2013).</li><li>Foil, L. D., 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=Factors+that+influence+the+prevalence+of+acaricide+resistance+and+tick-borne+diseases.">Factors that influence the prevalence of acaricide resistance and tick-borne diseases.</a> Vet. Parasitol. 125, 163-181 (2004).</li><li>Rodriguez, 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=High+level+expression+of+the+B.+microplus+Bm86+antigen+in+the+yeast+Pichia+pastoris+forming+highly+immunogenic+particles+for+cattle.">High level expression of the B. microplus Bm86 antigen in the yeast Pichia pastoris forming highly immunogenic particles for cattle.</a> J Biotechnol. 33, 135-146 (1994).</li><li>Rodriguez, 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=Effect+of+vaccination+with+a+recombinant+Bm86+antigen+preparation+on+natural+infestations+of+Boophilus+microplus+in+grazing+dairy+and+beef+pure+and+cross-bred+cattle+in+Brazil.">Effect of vaccination with a recombinant Bm86 antigen preparation on natural infestations of Boophilus microplus in grazing dairy and beef pure and cross-bred cattle in Brazil.</a> Vaccine. 13 (18), 1804-1808 (1995).</li><li>Lew-Tabor, A. E., Rodriguez Valle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+reverse+vaccinology+approaches+for+the+development+of+vaccines+against+ticks+and+tick+borne+diseases.">A review of reverse vaccinology approaches for the development of vaccines against ticks and tick borne diseases.</a> Ticks Tick Borne Dis. 7, 573-585 (2016).</li><li>Capella, A. N., Terra, W. R., Ribeiro, A. F., Ferreira, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeleton+removal+and+characterization+of+the+microvillar+membranes+isolated+from+two+midgut+regions+of+Spodoptera+frugiperda+(Lepidoptera).">Cytoskeleton removal and characterization of the microvillar membranes isolated from two midgut regions of Spodoptera frugiperda (Lepidoptera).</a> Insect Biochem. Mol. Biol. 27, 793-801 (1997).</li><li>Cioffi, M., Wolfersberger, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+separate+apical,+lateral+and+basal+plasma+membrane+from+cells+of+an+insect+epithelium.+A+procedure+based+on+tissue+organization+and+ultrastructure.">Isolation of separate apical, lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure.</a> Tissue Cell. 15, 781-803 (1983).</li><li>Koefoed, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+mechanical+method+to+isolate+the+basal+lamina+of+insect+midgut+epithelial+cells.">A simple mechanical method to isolate the basal lamina of insect midgut epithelial cells.</a> Tissue Cell. 17, 763-768 (1985).</li><li>Roche, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+purified+epithelial+cell+population+from+human+colon.">Isolation of a purified epithelial cell population from human colon.</a> Methods Mol. Med. 50, 15-20 (2001).</li><li>Terra, W. R., Costa, R. H., Ferreira, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membranes+from+insect+midgut+cells.">Plasma membranes from insect midgut cells.</a> An. Acad. Bras. Ci&#234;nc. 78, 255-269 (2006).</li><li>Vargas, A. E., Markoski, M. M., Ca&#241;edo, A. D., Helena, F., Nardi, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+isolation+and+culture+of+intestinal+epithelial+stem+cells+from+murine+intestine.">Identification, isolation and culture of intestinal epithelial stem cells from murine intestine.</a> Stem Cells. 879, 479-490 (2012).</li><li>Autengruber, A., Gereke, M., Hansen, G., Hennig, C., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+enzymatic+tissue+disintegration+on+the+level+of+surface+molecule+expression+and+immune+cell+function.">Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function.</a> Eur. J. Microbiol. Immunol. 2, 112-120 (2012).</li><li>Karhemo, P. 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=An+optimized+isolation+of+biotinylated+cell+surface+proteins+reveals+novel+players+in+cancer+metastasis.">An optimized isolation of biotinylated cell surface proteins reveals novel players in cancer metastasis.</a> J. Proteomics. 77, 87-100 (2012).</li><li>Obenchain, F. R., Galun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+Ticks.">Physiology of Ticks.</a> Current Themes in Tropical Science Volume 1. , 201-205 (1982).</li><li>Sonenshine, D., Roe, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3.1.">Chapter 3.1.</a> &amp;#34;Biology of Ticks&amp;#34. 1, (2014).</li><li>Raikhel, A. S., Balashov, Y. 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The authors have nothing to disclose.

Cattle tick infestations constitute a major problem for the cattle industry in tropical and subtropical regions of the world, with the most common method of control reliant upon the use of acaricides1,4. Bm86 was previously identified within the tick gut epithelial surface as a protective antigen against R. microplus infestation10, with limited success as a vaccine strategy due to Bm86 geographic sequence variation and the require...

Rhipicephalus microplus, the cattle tick, is the most significant ectoparasite in terms of economic impact on the cattle industry of tropical and sub-tropical regions as it vectors bovine tick fever (babesiosis), anaplasmosis and equine piroplasmosis1,2,3,4. Efforts have been dedicated to cattle tick control, to diminish the deleterious effect, however conventional methods such as the use of chemical acaricides have implicit drawbacks, such as the presence of chemical residues in milk and meat, and the increase in prevalence of chemically resistant ticks5,6,7. Consequently, the development of alternative methods of tick control have been studied, such as the use of natural resistance cattle, biological control (biopesticides) and vaccines4,5,6,7,8,9.
In the pursuit of proteins capable of being utilized as vaccine candidates, current research is focused upon the tick gut. The midgut wall is built from a single layer of epithelial cells resting on a thin basal lamina, with the outside of the basal lamina forming a network of muscle. Light and electron microscope observations indicate that the midgut consists of three types of cells: reserve (undifferentiated), secretory, and digestive. The number of cell types varies considerably depending upon the physiological phase. Secretory and digestive cells both originate from reserve cells18,19,20.
The construction of cDNA libraries to examine the composition of the tick gut has led to the identification of antigenic proteins, such as Bm86, as potential vaccine candidates2,3,4. The glycoprotein Bm86 is localized at the surface of tick gut cells and induces a protective immune response against the cattle tick (R. microplus) in vaccinated cattle. Anti-Bm86 IgGs produced by the immunized host are ingested by the tick, recognize this antigen on the surface of tick gut cells, and subsequently disturb tick gut tissue function and integrity. Vaccines based upon Bm86 antigens have shown effective control of R. microplus and Rhipicephalus annulatus, by reducing the number, weight and reproductive capacity of engorging females, resulting in a reduced larval infestation in subsequent tick generations4. However, Bm86 based vaccines are not effective against all tick stages and have demonstrated unsatisfactory efficacy against some geographical strains of R. microplus, consequently the beef and dairy industries have poorly adopted these vaccines2,4.
The ability to isolate epithelial cells from the tick gut is a significant innovation which would enable the progression of research to determine protein membrane composition including morphology and physiology under different environmental conditions. The method described here utilizes the chelating agent ethylenediaminetetraacetic acid (EDTA) and the reducing agent tris(2-carboxyethyl)phosphine (TCEP) to release the epithelium from its sub-epithelial support tissues10. The epithelium is recovered following mechanical disruption of the tissues by shaking, followed by discontinuous gradient centrifugation in Percoll. This paper describes a feasible and novel technique for the isolation of tick gut epithelial cells. Biotinylated cell surface proteins, isolated from the surface of these epithelial cells can subsequently analyzed in downstream applications such as FACS and/or LC-MS/MS-analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4% Trypan Blue</td>
			<td>ThermoFisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>3322</td>
			<td></td>
		</tr>
		<tr>
			<td>100mM Carbonate Buffer</td>
			<td></td>
			<td></td>
			<td>3.03 g Na2CO3, 6.0 g NaHCO3 1000 ml distilled water pH 9.6</td>
		</tr>
		<tr>
			<td>16 mL centrifuge tubes with sealing cap</td>
			<td>Thermo Scientific</td>
			<td>3138-0016</td>
			<td>Cool in ice prior to gradient</td>
		</tr>
		<tr>
			<td>250 &#181;M cell strainer</td>
			<td>Thermo Fisher</td>
			<td>87791</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA</td>
			<td>Sigma</td>
			<td>T0440</td>
			<td>Stored at 4C</td>
		</tr>
		<tr>
			<td>30% Hydrogen Peroxide</td>
			<td>Labscene</td>
			<td>BSPA5.500</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-MOPS Gel</td>
			<td>Gen Script</td>
			<td>M42015</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Chloro-1-naphthol tablet</td>
			<td>Sigma-Aldrich</td>
			<td>C6788</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon Tube</td>
			<td>Corning Blue</td>
			<td></td>
			<td>30 x 115mm style. Polyproplyene conical tube.</td>
		</tr>
		<tr>
			<td>70 &#181;M cell strainer</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>AP15 filter paper</td>
			<td>Millipore</td>
			<td>AO1504200</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin (Type A) Conjugation Kit</td>
			<td>Abcam</td>
			<td>Ab102865</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td></td>
		</tr>
		<tr>
			<td>DP Manager&#160;</td>
			<td>Olympus</td>
			<td>2.2.1.195</td>
			<td>Cell imagery software</td>
		</tr>
		<tr>
			<td>Duct Tape</td>
			<td>Home Handyman</td>
			<td></td>
			<td>48mm x 25mm Duct Tape</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td>DMEM - ice cold for protocol</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Amresco</td>
			<td>0105-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>F96 Maxisorp Immuno Plate</td>
			<td>Nunc</td>
			<td>439454</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12003C</td>
			<td>FCS</td>
		</tr>
		<tr>
			<td>Fluorescence microscope&#160;</td>
			<td>&#160;Olympus&#160;</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>F6057-20ML</td>
			<td>DAPI</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td></td>
			<td>#9 Dumont - Switerzland</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Glycerol for molecular biology &#62;99%</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>410225</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand-Held Counter</td>
			<td>Officeworks</td>
			<td>JA0376230</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Sigma Life Sciences</td>
			<td>H9394</td>
			<td>HBSS &#8211; ice cold for protocol</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Optik Lakor</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>L-Glutathione oxidized</td>
			<td>Sigma-Aldrich</td>
			<td>G4376</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Separation Stand</td>
			<td>Novagen</td>
			<td>-</td>
			<td>4-Tube Magnetic Separation Rack</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>179337</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q Water</td>
			<td>Millipore</td>
			<td>ZRXQ003WW</td>
			<td>Integral Water Purification System for Ultrapure Water</td>
		</tr>
		<tr>
			<td>Nitrocellulose Membrane</td>
			<td>Life Sciences</td>
			<td>66485</td>
			<td>30cm x 3M pure nitrocellulose membrane</td>
		</tr>
		<tr>
			<td>PageRuler Prestained protein Ladder</td>
			<td>Thermo-Fisher</td>
			<td>SM0671</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>1.16 g Na2HPO4, 0.1 g KCl, 0.1 g K3PO4, 4.0 g NaCl (500 ml distilled water) pH 7.4</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Sigma-Aldrich</td>
			<td>P1644-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Masterflex</td>
			<td>7518-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>P6560</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Protein-Free T20 PBS Blocking Buffer</td>
			<td>Thermo-Scientific</td>
			<td>37573</td>
			<td>Stored at 4C. Blocking Buffer</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8215-5ML</td>
			<td>PIC &#8211; stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Quick Start Bradford Dye Reagent 1x</td>
			<td>Biorad</td>
			<td>500-0205</td>
			<td>For Bradford Assay</td>
		</tr>
		<tr>
			<td>Quick Start BSA Standards</td>
			<td>Biorad</td>
			<td>500-0207</td>
			<td>BSA standards for Bradford Assay</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Lab. Co</td>
			<td></td>
			<td>Size 11 Scalpel</td>
		</tr>
		<tr>
			<td>SilverQuest TM Staining Kit</td>
			<td>Invitrogen</td>
			<td>LC6070</td>
			<td></td>
		</tr>
		<tr>
			<td>Simply Blue TM Safe Stain&#160;</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall C6+ Ultracentrifuge</td>
			<td>Thermo Scientific</td>
			<td>46910</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin (HRP)</td>
			<td>Abcam</td>
			<td>AB7403</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Magnetic Beads</td>
			<td>New England Biolabs</td>
			<td>S1420S</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Glue - Ultra Fast Mini</td>
			<td>UHU</td>
			<td></td>
			<td>UHU Super Glue 1mg. Ultra Fast mini</td>
		</tr>
		<tr>
			<td>Table-top Centrifuge</td>
			<td>Eppendorf</td>
			<td>22331</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Thermo Fisher</td>
			<td>20490</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Biorad</td>
			<td>161-0407</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P2287-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Ratek</td>
			<td>VM1</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Grant</td>
			<td>GD100</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of biotinylated cell surface proteins from rhipicephalus microplus epithelial gut cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Epithelial cells were isolated from the gut tissues of R. microplus as per the schematic presented in Figure 1. Representative fluorescence microscopy imagery of tick gut epithelial cells prepared using this protocol are shown in Figure 2A and 2B. As the cell isolation is conducted upon semi-engorged R. microplus, cells appear as singular, spher...

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Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Purification of biotinylated cell surface protein from Rhipicephalus microplus epithelial gut cells. Within this study, a modified Percoll gradient-based methodology was utilized to isolate individual epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for the utilization in downstream applications.Dissection of the gut epithelium from semi-engorged Rhipicephalus microplus. You will need the following materials. Adhere a strip of duct tape to the bottom of a Petri dish.Add a drop of Super Glue to the tape. Place the tick ventral side down on the Super Glue, allowing it to dry. Completely submerge the tick in 100 mL of PBS.Utilizing a size 11 scalpel, cut from the top of the eyes of to the bottom festoons on both sides of the tick. Using sterile forceps, completely remove the scutum and alloscutum to expose the internal organs. Remove the fine white threadlike organs and other membranes.Remove the gut using forceps. Store gut in ice-cold HBSS and PIC. Epithelial cell disassociation.You will need the following materials. Pour the dissected gut onto a 70 micron cell strainer inside a 50 mL Falcon tube. As a vacuum may form within the Falcon tube, lift the filter to allow for air circulation.Flush the gut tissue with 50 mL ice-cold HBSS with PIC. Resuspend guts in 30 mL ice-cold HBSS with PIC. If necessary, use a sterile scraper to assist in gut resuspension.Centrifuge at 500 g. Remove the supernatant and repeat the wash process three times. Resuspend the gut cells in 10 mL of DMEM, 2%FCS, 5 millimolar EDTA, one millimolar TCEP and PIC.Mix gently. Incubate for 60 minutes at 37 degrees Celsius under slow rotation using a roller. Filter the suspension through a 250 micron cell strainer.Vortex the flow through. And filter through a 70 micron cell strainer, collecting the remaining flow through. Centrifuge the suspension.Isolation of single epithelial cells using a Percoll gradient. You will need the following materials. By filtering through AP15 pre-filter paper, prepare 40%and 20%Percoll in Milli-Q water.Load a sterile syringe with the Percoll solution. Apply it to the filter rig and slowly discharge the Percoll. Cool at four degrees Celsius for an hour prior to layering the gradient.Set up the peristaltic pump as shown. Using a peristaltic pump set at the lowest speed, layer three mL of 40%Percoll into a 16 mL ultracentrifuge tube. Allow it to settle on ice for 15 minutes.Speed of the pump should lead to a less than one mL per minute flow rate. Tilting the tube to a 45 degree angle, use the peristaltic pump to layer the 20%Percoll on top of the 40%layer. Ensure that the output tube is resting against the centrifuge tube to prevent droplets mixing with lower layers.Speed of the pump should lead to less than one mL per minute flow rate. Allow the layers to settle on ice for 15 minutes. Using the peristaltic pump at less than one mL per minute flow rate to layer three mL of DMEM solution containing the isolated epithelial cells over the 20 to 40%Percoll gradient.Ensure the output tube is resting against the centrifuge tube to prevent droplets mixing with the lower layers, disrupting the gradient. Centrifuge at 600 g for 10 minutes. Collect interphases between the DMEM, 20%Percoll gradient, and the 20%40%Percoll gradients to isolate epithelial single cells.Cell surface protein biotinylation and the isolation of biotinylated surface proteins. You will need the following materials. Biotinylate 100 microliters of single-cell epithelial cells using Biotin Type A Conjugation Kit as per manufacturer's instructions.Add 100 microliters of PBS, 1%Triton X-100, 10%glycerol, 100 micromolar oxidized glutathione, and PIC to the biotinylated cells. Incubate on ice for an hour with gentle mixing every 10 minutes. Centrifuge the cell extract at 20, 000 g at four degrees Celsius for 20 minutes and set aside.Prepare 50 microliters of streptavidin magnetic beads by washing with 1, 000 microliters of TBS 1%Tween-20. Mix gently by flicking with your finger. Place the tube into a magnetic stand, collecting the beads against the side of the tube.Remove and discard the supernatant. Combine 300 microliters of biotinylated cell surface proteins with washed magnetic beads. Incubate for two hours at room temperature with agitation.Collect the beads with the magnetic stand, remove, and discard the supernatant. Add 300 microliters of TBS 1%Tween-20 to the tube, gently mixing to resuspend the beads. Collect beads, remove, and discard the supernatant.Repeat this wash step twice. Add 100 microliters of the 1 molar glycine pH two to the magnetic beads. And incubate at room temperature for five minutes.Collect beads and remove supernatant containing eluted biotinylated surface proteins. Figure one is a representation of the protocol utilized to isolate epithelial cells and their surface proteins from a whole tick. Utilizing this protocol, approximately 1.2 by 10 to the seven cells per mL with 75 to 80%viability and 20 to 24 micrograms of purified biotinylated surface proteins can be purified from an initial dissection of 50 ticks.Representative fluorescence microscopy imagery of tick get epithelial cells prepared using this protocol are shown in figure 2A and 2B. Cells appear as singular, spherical, smooth surface morphology and a consistent size throughout the sample. Poor isolations are visualized to contain incomplete dissociation of individual epithelial cells with varying cell populations identified through the varying cell sizes and morphologies.Cross-contamination from host proteins, figure 3A, can be minimized by adequately rinsing tick guts to produce a white or clear Percoll gradient, figure 3B. Comparison of proteins by SDS-PAGE, figure 4A, silver stain, figure 4B, dot blot, figure five, and ELISA, figure six, indicates that the described methodologies successfully purified biotinylated surface proteins from individual epithelial cells isolated from Rhipicephalus microplus tick guts. In conclusion, the methods utilized in this study successfully isolated individual epithelial cells from the whole gut of Rhipicephalus microplus.Proteins from the surface of Rhipicephalus microplus epithelial cells were obtained for further analysis and studies. Finally, the protocol developed has demonstrated the potential yield of individual epithelial cells from the tick gut and the efficiency of biotinylated surface proteins of the cell. The protocol developed can be utilized in any tick species of economic significance in an effort to investigate tick-host interactions by studying the membrane protein composition of the gut.

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9.3K visualizações.  The University of Queensland. Purification of biotinylated cell surface protein from Rhipicephalus microplus epithelial gut cells. Within this study, a modified Percoll gradient-based methodology was utilized to isolate individual epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for the utilization in downstream applications.Dissection of the gut epithelium from semi-engorged Rhipicephalus microplus. You will need the...