Name: detecting wolbachia strain walbb in aedes albopictus cell lines
Uploaded: 2022-06-01T15:00:00
Description: Watch this Scientific Journal Video about Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines at JoVE.com

We thank Dr. Xin-Ru Wang from the University of Minnesota for insightful suggestions and guidance. This work was supported by a grant from the National Natural Science Foundation of China (No.81760374).

1. Materials and reagents
<ol>
	<li>Use pyrogen-free solutions and media for cell culture (see the Table of Materials).</li>
	<li>Use ultrapure water to prepare all solutions.</li>
	<li>Be careful in selecting fetal bovine serum (FBS) for cell culture, following a lot-check process. 
	NOTE: As FBS lots are subject to regular change, it is impossible to state the relevant catalog and lot numbers in this protocol.</li>
	<li>Select Aa23 cell strains derived from A. albopictus</em...

<ol>
	<li>Wiwatanaratanabutr, I., Kittayapong, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+crowding+and+temperature+on+Wolbachia+infection+density+among+life+cycle+stages+of+Aedes+albopictus.">Effects of crowding and temperature on Wolbachia infection density among life cycle stages of Aedes albopictus.</a> Journal of Invertebrate Patholology. 102 (3), 220-224 (2009).</li><li>Sinkins, S. P., Braig, H. R., O'Neill, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia+superinfections+and+the+expression+of+cytoplasmic+incompatibility.">Wolbachia superinfections and the expression of cytoplasmic incompatibility.</a> Proceedings of Biologial Sciences. 261 (1362), 325-330 (1995).</li><li>Dobson, S. 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=Wolbachia+infections+are+distributed+throughout+insect+somatic+and+germ+line+tissues.">Wolbachia infections are distributed throughout insect somatic and germ line tissues.</a> Insect Biochemistry and Molecular Biology. 29 (2), 153-160 (1999).</li><li>O'Neill, S. 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=In+vitro+cultivation+of+Wolbachia+pipientis+in+an+Aedes+albopictus+cell+line.">In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line.</a> Insect Molecular Biology. 6 (1), 33-39 (1997).</li><li>Sinha, A., Li, Z., Sun, L., Carlow, C. K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+genome+sequence+of+the+Wolbachia+wAlbB+endosymbiont+of+Aedes+albopictus.">Complete genome sequence of the Wolbachia wAlbB endosymbiont of Aedes albopictus.</a> Genome Biology and Evoution. 11 (3), 706-720 (2019).</li><li>Sinkins, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia+and+cytoplasmic+incompatibility+in+mosquitoes.">Wolbachia and cytoplasmic incompatibility in mosquitoes.</a> Insect Biochemistry and Molecular Biology. 34 (7), 723-729 (2004).</li><li>Fallon, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+properties+of+an+Aedes+albopictus+mosquito+cell+line+infected+with+Wolbachia+strain+wAlbB.">Cytological properties of an Aedes albopictus mosquito cell line infected with Wolbachia strain wAlbB.</a> In Vitro Cellular Developmental Biology - Animals. 44 (5-6), 154-161 (2008).</li><li>Hilgenboecker, K., Hammerstein, P., Schlattmann, P., Telschow, A., Werren, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+many+species+are+infected+with+Wolbachia?-A+statistical+analysis+of+current+data.">How many species are infected with Wolbachia?-A statistical analysis of current data.</a> Microbiology Letters. 281 (2), 215-220 (2008).</li><li>Werren, J. H., Baldo, L., Clark, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia:+master+manipulators+of+invertebrate+biology.">Wolbachia: master manipulators of invertebrate biology.</a> National Review of Microbiology. 6 (10), 741-751 (2008).</li><li>Kittayapong, P., Baisley, K. J., Baimai, V., O'Neill, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+diversity+of+Wolbachia+infections+in+Southeast+Asian+mosquitoes+(Diptera:+Culicidae).">Distribution and diversity of Wolbachia infections in Southeast Asian mosquitoes (Diptera: Culicidae).</a> Journal of Medical Entomology. 37 (3), 340-345 (2000).</li><li>O'Neill, S. L., Hoffmann, A., Werren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Influential passengers: inherited microorganisms and arthropod reproduction. , (1997).</li><li>McGraw, E. A., O'Neill, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+insecticides:+new+thinking+on+an+ancient+problem.">Beyond insecticides: new thinking on an ancient problem.</a> National Review of Microbiology. 11 (3), 181-193 (2013).</li><li>Bourtzis, K., 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=Harnessing+mosquito-Wolbachia+symbiosis+for+vector+and+disease+control.">Harnessing mosquito-Wolbachia symbiosis for vector and disease control.</a> Acta Tropica. 132, 150-163 (2014).</li><li>Turelli, M., Hoffmann, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+spread+of+an+inherited+incompatibility+factor+in+California+Drosophila.">Rapid spread of an inherited incompatibility factor in California Drosophila.</a> Nature. 353 (6343), 440-442 (1991).</li><li>Hoffmann, A. A., 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=Successful+establishment+of+Wolbachia+in+Aedes+populations+to+suppress+dengue+transmission.">Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission.</a> Nature. 476 (7361), 454-457 (2011).</li><li>Walker, T., 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=The+wMel+Wolbachia+strain+blocks+dengue+and+invades+caged+Aedes+aegypti+populations.">The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations.</a> Nature. 476 (7361), 450-453 (2011).</li><li>Hughes, G. L., Koga, R., Xue, P., Fukatsu, T., Rasgon, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia+infections+are+virulent+and+inhibit+the+human+malaria+parasite+Plasmodium+falciparum+in+Anopheles+gambiae.">Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae.</a> PLoS Pathogens. 7 (5), 1002043 (2011).</li><li>Bian, G., Xu, Y., Lu, P., Xie, Y., Xi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endosymbiotic+bacterium+Wolbachia+induces+resistance+to+dengue+virus+in+Aedes+aegypti.">The endosymbiotic bacterium Wolbachia induces resistance to dengue virus in Aedes aegypti.</a> PLoS Pathogens. 6 (4), 1000833 (2010).</li><li>Moreira, L. A., 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+Wolbachia+symbiont+in+Aedes+aegypti+limits+infection+with+dengue,+Chikungunya,+and+Plasmodium.">A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium.</a> Cell. 139 (7), 1268-1278 (2009).</li><li>Caragata, E. 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=Dietary+cholesterol+modulates+pathogen+blocking+by+Wolbachia.">Dietary cholesterol modulates pathogen blocking by Wolbachia.</a> PLoS Pathogens. 9 (6), 1003459 (2013).</li><li>Zhang, G., Hussain, M., O'Neill, S. L., Asgari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia+uses+a+host+microRNA+to+regulate+transcripts+of+a+methyltransferase,+contributing+to+dengue+virus+inhibition+in+Aedes+aegypti.">Wolbachia uses a host microRNA to regulate transcripts of a methyltransferase, contributing to dengue virus inhibition in Aedes aegypti.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (25), 10276-10281 (2013).</li><li>Tortosa, P., Courtiol, A., Moutailler, S., Failloux, A. B., Weill, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chikungunya-Wolbachia+interplay+in+Aedes+albopictus.">Chikungunya-Wolbachia interplay in Aedes albopictus.</a> Insect Molecular Biology. 16 (7), 677-684 (2008).</li><li>Lu, P., Bian, G., Pan, X., Xi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia+induces+density-dependent+inhibition+to+dengue+virus+in+mosquito+cells.">Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells.</a> PLoS Neglected Tropical Diseases. 6 (7), 1754 (2012).</li><li>Ghosh, A., Jasperson, D., Cohnstaedt, L. W., Brelsfoard, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+Culicoides+sonorensis+biting+midge+cell+lines+with+Wolbachia+pipientis.">Transfection of Culicoides sonorensis biting midge cell lines with Wolbachia pipientis.</a> Parasite Vectors. 12 (1), 483 (2019).</li><li>Zhou, W., Rousset, F., O'Neill, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+and+PCR-based+classification+of+Wolbachia+strains+using+wsp+gene+sequences.">Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences.</a> The Royal Society Publishing. Proceedings B. 265 (1395), 509-515 (1998).</li><li>Park, M. S., Takeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+PaAtg8+and+roles+of+autophagy+in+adaptation+to+starvation+with+respect+to+the+fat+body+and+midgut+of+the+Americana+cockroach,+Periplaneta+americana.">Cloning of PaAtg8 and roles of autophagy in adaptation to starvation with respect to the fat body and midgut of the Americana cockroach, Periplaneta americana.</a> Cell Tissue Research. 356 (2), 405-416 (2014).</li><li>Geng, S. C., Li, X. L., Fang, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+circovirus+3+capsid+protein+induces+autophagy+in+HEK293T+cells+by+inhibiting+phosphorylation+of+the+mammalian+target+of+rapamycin.">Porcine circovirus 3 capsid protein induces autophagy in HEK293T cells by inhibiting phosphorylation of the mammalian target of rapamycin.</a> Journal of Zhejiang University Science B. 21 (7), 560-570 (2020).</li><li>Taylor, S. 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Detection of intracellular Wolbachia infection is essential for the study of Wolbachia-host interactions and the confirmation of successful transfection of cells with novel strains. In this protocol, four methods were used to successfully detect intracellular Wolbachia infection at the nucleic acid and protein levels. These four experimental methods corroborated and improved the detection accuracy of Wolbachia infection of cells. 
PCR was used to detect the ...

The Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae), which is a key vector of dengue virus (DENV) in Asia and other parts of the world1, is a natural host of two types of the intracellular bacteria, Wolbachia (wAlbA and wAlbB), which are distributed throughout the germ line and somatic tissue2,3. The Aa23 cell line derived from A. albopictus embryos&#160;consists of at least two morphological cell types, both of which support infection4 and may be cured of native Wolbachia infection using antibiotics (Aa23-T). Given that Aa23 retains only wAlbB, it is a useful model for the study of host-endosymbiont interactions4,5,6.
Wolbachia is maternally transmitted and infects an estimated 65% of insect species8,9 and 28% of mosquito species10. It infects a variety of tissues and forms an intimate symbiotic relationship with the host, usually inducing cytoplasmic incompatibility (CI)11 and population replacement by manipulating the host reproductive system12,13. These host responses have been observed in natural populations of Drosophila simulans14 and in A. aegypti in a laboratory cage and field trial15. An important nonreproductive manipulation elicited by Wolbachia is induced host resistance to a variety of pathogens, including DENV, Chikungunya virus (CHIKV), and West Nile virus (WNV)16,17, that may be mediated by an improved innate immune system of the symbiont18,19, competition between Wolbachia and viruses for essential host resources20, and manipulation of host viral defense pathways21.
This protocol has been developed for studying these underlying mechanisms of Wolbachia-induced host antiviral responses. It uses four methods of detection of intracellular Wolbachia infection of Aa23 cells. These methods provide a strong theoretical basis for studies of intracellular Wolbachia infection of other host species. The first method, PCR-a powerful technique allowing the enzymatic amplification of specific regions of DNA without utilizing conventional cloning procedures-was used to detect Wolbachia DNA and determine the presence/absence of Wolbachia infection22. The second method measures Wolbachia DNA copy density using quantitative PCR (qPCR) for reliable detection and measurement of products generated during each PCR cycle that is directly proportionate to the amount of template before PCR23. The third method detects the presence of intracellular Wolbachia proteins, using western blot-one of the most powerful tools for detecting specific proteins in complex mixtures by combining the high separation power of electrophoresis, the specificity of antibodies, and the sensitivity of chromogenic enzymatic reactions. The final method is an immunofluorescence assay (IFA) that combines immunology, biochemistry, and microscopy to detect the Wolbachia surface protein (wsp) through an antigen-antibody reaction to confirm the cellular uptake of Wolbachia and determine its cellular localization.
This paper describes the four methods listed above to verify the existence of Wolbachia in the cells, which can be used to detect whether the exogenous Wolbachia was successfully transfected and the Wolbachia in the cell was cleared. After determining whether Wolbachia is present in the cells or not, a variety of different analyses can be performed, including genomics, proteomics, or metabolomics. This protocol demonstrates the detection of Wolbachia through Aa23 cells but can also be used in other cells.

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		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>SteREO Discovery V8</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scietific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Pipetman</td>
			<td>F167380</td>
			<td>P10</td>
		</tr>
		<tr>
			<td>inSituX platform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>In-house developed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cerium doped yttrium aluminum garnet</td>
			<td>MSE Supplies</td>
			<td></td>
			<td>Ce:Y3Al5O12, YAG single crystal substrates</td>
		</tr>
		<tr>
			<td>Chip holder</td>
			<td>In-house developed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Control software</td>
			<td>In-house developed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil</td>
			<td>Cargille Laboratories</td>
			<td>16482</td>
			<td>Type A low viscosity 150 cSt</td>
		</tr>
		<tr>
			<td>inSituX platform</td>
			<td>In-house developed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IR light source&#160;</td>
			<td>Thorlabs Incorporated</td>
			<td>LED1085L</td>
			<td>LED with a Glass Lens, 1085 nm, 5 mW, TO-18</td>
		</tr>
		<tr>
			<td>Outer ring&#160;</td>
			<td>In-house developed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pump lasers&#160;</td>
			<td>Thorlabs Incorporated</td>
			<td>LD785-SE400</td>
			<td>785 nm, 400 mW, &#216;9 mm, E Pin Code, Laser Diode</td>
		</tr>
		<tr>
			<td>Raspberry Pi</td>
			<td>Raspberry Pi Fundation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Retaining ring</td>
			<td>Thorlabs Incorporated</td>
			<td>SM1RR</td>
			<td>SM1 retaining ring for &#216;1&#34; lens tubes and mounts</td>
		</tr>
		<tr>
			<td>Seedless quartz crystal</td>
			<td>University Wafers, Inc.</td>
			<td>U01-W2-L-190514</td>
			<td>25.4 mm diameter Z-cut 0.05 mm thickness double side polish 8 mm on -X</td>
		</tr>
		<tr>
			<td>Shim</td>
			<td>In-house developed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray beam stop</td>
			<td>In-house developed</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting wolbachia strain walbb in aedes albopictus cell lines

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation of RNA virus transmission using Wolbachia-transfected mosquitoes. Key strategies to control viruses include the manipulation of host reproduction via cytoplasmic incompatibility and the inhibition of viral transcripts via immune priming and competition for host-derived resources. However, the underlying mechanisms of the responses of Wolbachia-transfected mosquitoes to viral infection are poorly understood. This paper presents a protocol for the in vitro identification of Wolbachia infection at the nucleic acid and protein levels in Aedes albopictus (Diptera: Culicidae) Aa23 cells to enhance the understanding of the interactions between Wolbachia and its insect vectors. Through the combined use of polymerase chain reaction (PCR), quantitative PCR, western blot, and immunological analytical methods, a standard morphologic protocol has been described for the detection of Wolbachia-infected cells that is more accurate than the use of a single method. This approach may also be applied to the detection of Wolbachia infection in other insect taxa.

Before Wolbachia was detected, Aa23 and Aa23-T cells were observed under a light microscope to determine any morphological differences between the two cell lines. Aa23 and Aa23-T cells have at least two cell morphologies but no obvious morphological difference between the two cell types (Figure 1). Here, Aa23 cells were used as a model system to detect Wolbachia infection using four methods. Positive amplification of the Wolbachiawsp gene using the...

Watch this Scientific Journal Video about Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines at JoVE.com

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

Four methods were used to detect intracellular Wolbachia, which complemented each other and improved the detection accuracy of Wolbachia infection of Aedes albopictus-derived Aa23 and Aa23-T&#160;cured of native Wolbachia infection using antibiotics.

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation ...

This protocol helps verify the existence of Wolbachia in cells, which is the basis of understanding the interaction between Wolbachia and its host. In this protocol, four methods were used to successfully detect intracellular Wolbachia infection, which corroborated and improved the detection accuracy of Wolbachia infection of cells. Demonstrating the procedure will be Qiuqiu Xiao, a postgraduate from the Key Laboratory of Modern Pathogen Biology and Characteristics.Begin by observing unstained Aa23 and Aa23-T cells at 100x magnification using light microscopy. Culture the cells for five to seven days to reach 70 to 80%confluence per flask. Using a pipette, transfer 1 mL of the culture medium to a microcentrifuge tube.Centrifuge at 100 RCF for five minutes to harvest the cells. Remove the supernatant and store the resulting cell pellet at minus 20 degrees Celsius. Culture the cells for five to seven days to reach 90 to 100%confluence per flask.Resuspend the pellets in 0.40 mL of PBS and transfer 50 microliters of this solution to a microcentrifuge tube. Add five microliters of 20 mg/mL proteinase K and incubate at 55 degrees Celsius for one hour. Then incubate the tubes at 99 degrees Celsius for 15 minutes to obtain the crude DNA and store it at minus 20 degrees Celsius.To perform PCR, prepare a total reaction mixture of 20 microliters and set the PCR conditions as described in the text manuscript. Detect the DNA on a 1%agarose gel using a gel imager. Perform quantitative PCR amplification using TB Green in a real-time PCR system and record the results of each reaction.Analyze the DNA in a total reaction volume of 20 microliters and set the quantitative PCR conditions as described in the text manuscript. Calculate the WSP and RPS6 gene copy numbers in the cells according to the established standard curve. Then calculate the Wolbachia relative density by the copy number of wsp/RPS6.Analyze the data using an independent samples t-test. Add 200 microliters of lysis buffer to the cell pellet and incubate on ice for 30 minutes. Centrifuge the lysates at 12, 000 RCF for 10 minutes at four degrees Celsius and aspirate the supernatant.Analyze the wsp concentration of the supernatant using a BCA protein assay kit, following the manufacturer's instructions. Load equal amounts of protein on a 15%SDS-PAGE gel. Transfer the protein to nitrocellulose membranes and block the membranes with 5%skimmed milk for two hours at room temperature.Then wash the membranes three times with TBST. Incubate the membranes overnight at 4 degrees Celsius with 100 microliters of anti-wsp polyclonal antibody prepared by diluting the primary antibody with 5%skimmed milk. Wash the primary antibody three times with TBST.Add 100 microliters of horseradish peroxidase-conjugated secondary antibody to the membranes and incubate it on a shaker for one hour at room temperature. Wash the membranes thrice with TBST and mix 20 microliters of solution A and 20 microliters of solution B in the chemiluminescence detection kit at a ratio of one to one. Immerse the entire membrane into the luminescence solution simultaneously and observe the signals using a multifunctional imaging system.On a Laser confocal Petri dish, incubate Aa23 and Aa23-T cells for three to four days at 28 degrees Celsius under 5%atmospheric carbon dioxide. When the cell reaches 60 to 70%confluence, wash the cells three times with PBS. Fix the cells in one mL of 4%paraformaldehyde for 20 minutes at four degrees Celsius.Wash the fixed cells using PBST for five minutes and rewash the cells twice with PBS. Incubate the Petri dish in one mL of 3%bovine serum albumin for one hour at 37 degrees Celsius. Add 100 microliters of mouse anti-wsp polyclonal antibody and mouse serum to the slides and incubate it overnight in a wet box at four degrees Celsius.Remove the Petri dish from the wet box and return them to room temperature. Wash them three times with PBS and remove the PBS. Protect the following procedures from light.Incubate the Petri dish with 100 microliters of an Alexa Fluor 488-conjugated goat anti-mouse antibody at 37 degrees Celsius for 30 minutes. Incubate the sample Petri dish with 50 microliters of DAPI diluted in PBS to 10 micrograms/mL at 37 degrees Celsius for five minutes and then wash them three times with PBS. Observe the immunostaining under a confocal microscope.Before detecting Wolbachia, Aa23 and Aa23-T cells were observed under a light microscope to determine morphological differences between the two cell lines. The Aa23 and AA23-T cells had at least two cell morphologies but no apparent morphological differences were observed between the two cell types. Using the diagnostic primers 81F/691R, positive amplification of the Wolbachia wsp gene was detected in Aa23 cells but not in Aa23-T cells.The analysis of Wolbachia density in the cell lines showed a wsp/rps6 ratio of 2.4 in Aa23 cells but no Wolbachia in Aa23-T cells. Western blot analysis of protein extracts showed a strong wsp signal for Aa23 but no signal was detected for Aa23-T. The indirect immunofluorescence assay detected the wsp protein in Aa23 cells, while only DAPI-stained DNA was detected in Aa23-T cells.Ensure that the culture flasks are incubated at 28 degrees Celsius under 5%atmospheric carbon dioxide for five to seven days. This procedure can be followed by electron microscopy where one needs to pay particular attention to sample preparation and sectioning. The four experimental methods used in this study confirmed the detection accuracy of Wolbachia infection in cells, which is also applicable to other types of cells and tissues.

Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

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