Name: collection, isolation, and flow cytometric analysis of human endocervical samples
Uploaded: 2014-07-06T15:00:00
Description: Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

Ethics statement: The research ethics boards of both the University of Manitoba and Kenyatta National Hospital/University of Nairobi approved this study and written informed consent was obtained from all study participants.
1. Preparation of Media and CMC Collection Tubes
<ol>
	<li>Prepare phosphate buffered saline (PBS) solution (137.93 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4). Autoclave for sterility. This can be stored at 4 &#186;C ...

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNAIDS.+Global+Report+2013.">UNAIDS. Global Report 2013.</a> http://www.unaids.org/en/media/unaids/contentassets/documents/epidemiology/2013/gr2013/UNAIDS_Global_Report_2013_en.pdf. , (2013).</li><li>Rodriguez-Garcia, M., Patel, M. V., Wira, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+and+adaptive+anti-HIV+immune+responses+in+the+female+reproductive+tract.">Innate and adaptive anti-HIV immune responses in the female reproductive tract.</a> Journal of reproductive immunology. 97, 74-84 (2013).</li><li>Douek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+disease+progression:+immune+activation,+microbes,+and+a+leaky+gut.">HIV disease progression: immune activation, microbes, and a leaky gut.</a> Topics in HIV medicine : a publication of the International AIDS Society, USA. 15, 114-117 (2007).</li><li>Hofer, U., Speck, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbance+of+the+gut-associated+lymphoid+tissue+is+associated+with+disease+progression+in+chronic+HIV+infection.">Disturbance of the gut-associated lymphoid tissue is associated with disease progression in chronic HIV infection.</a> Seminars in immunopathology. 31, 257-266 (2009).</li><li>Kaushic, C., Ferreira, V. H., Kafka, J. K., Nazli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+infection+in+the+female+genital+tract:+discrete+influence+of+the+local+mucosal+microenvironment.">HIV infection in the female genital tract: discrete influence of the local mucosal microenvironment.</a> American journal of reproductive immunology. 63, 566-575 (2010).</li><li>Hladik, F., Hope, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+infection+of+the+genital+mucosa+in+women.">HIV infection of the genital mucosa in women.</a> Current HIV/AIDS reports. 6, 20-28 (2009).</li><li>Sharkey, D. J., Tremellen, K. P., Jasper, M. J., Gemzell-Danielsson, K., Robertson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seminal+fluid+induces+leukocyte+recruitment+and+cytokine+and+chemokine+mRNA+expression+in+the+human+cervix+after+coitus.">Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus.</a> Journal of immunology. 188, 2445-2454 (2012).</li><li>Saba, E., 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=Productive+HIV-1+infection+of+human+cervical+tissue+ex+vivo+is+associated+with+the+secretory+phase+of+the+menstrual+cycle.">Productive HIV-1 infection of human cervical tissue ex vivo is associated with the secretory phase of the menstrual cycle.</a> Mucosal immunology. 6, 1081-1090 (2013).</li><li>St John, E. P., Martinson, J., Simoes, J. A., Landay, A. L., Spear, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+activation+and+maturation+induced+by+mucosal+fluid+from+women+with+bacterial+vaginosis.">Dendritic cell activation and maturation induced by mucosal fluid from women with bacterial vaginosis.</a> Clinical immunology. 125, 95-102 (2007).</li><li>Liebenberg, L. J., 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=Stability+and+transport+of+cervical+cytobrushes+for+isolation+of+mononuclear+cells+from+the+female+genital+tract.">Stability and transport of cervical cytobrushes for isolation of mononuclear cells from the female genital tract.</a> Journal of immunological methods. 367, 47-55 (2011).</li><li>Hirbod, T., Kaldensjo, T., Broliden, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+distribution+of+HIV-binding+CCR5+and+C-type+lectin+receptors+in+the+human+endocervical+mucosa.">In situ distribution of HIV-binding CCR5 and C-type lectin receptors in the human endocervical mucosa.</a> PloS one. 6, (2011).</li><li>Hasselrot, 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=Feasibility+and+safety+of+cervical+biopsy+sampling+for+mucosal+immune+studies+in+female+sex+workers+from+Nairobi,+Kenya.">Feasibility and safety of cervical biopsy sampling for mucosal immune studies in female sex workers from Nairobi, Kenya.</a> PloS one. 7, (2012).</li><li>McKinnon, L. 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=Optimizing+viable+leukocyte+sampling+from+the+female+genital+tract+for+clinical+trials:+an+international+multi-site+study.">Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.</a> PloS one. 9, (2014).</li><li>Lajoie, J., 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+distinct+cytokine+and+chemokine+profile+at+the+genital+mucosa+is+associated+with+HIV-1+protection+among+HIV-exposed+seronegative+commercial+sex+workers.">A distinct cytokine and chemokine profile at the genital mucosa is associated with HIV-1 protection among HIV-exposed seronegative commercial sex workers.</a> Mucosal immunology. 5, (2012).</li><li>Burgener, 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=Comprehensive+Proteomic+Study+Identifies+Serpin+and+Cystatin+Antiproteases+as+Novel+Correlates+of+HIV-1+Resistance+in+the+Cervicovaginal+Mucosa+of+Female+Sex+Workers.">Comprehensive Proteomic Study Identifies Serpin and Cystatin Antiproteases as Novel Correlates of HIV-1 Resistance in the Cervicovaginal Mucosa of Female Sex Workers.</a> Journal of proteome research. 10, (2011).</li><li>Burgener, 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+systems+biology+examination+of+the+human+female+genital+tract+shows+compartmentalization+of+immune+factor+expression.">A systems biology examination of the human female genital tract shows compartmentalization of immune factor expression.</a> Journal of virology. 87, (2013).</li><li>Bere, A., Denny, L., Naicker, P., Burgers, W. A., Passmore, 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=HIV-specific+T+cell+responses+detected+in+the+genital+tract+of+chronically+HIV-infected+women+are+largely+monofunctional.">HIV-specific T cell responses detected in the genital tract of chronically HIV-infected women are largely monofunctional.</a> Immunology. 139, (2013).</li><li>McKinnon, L. 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=Characterization+of+a+Human+Cervical+CD4++T+Cell+Subset+Coexpressing+Multiple+Markers+of+HIV+Susceptibility.">Characterization of a Human Cervical CD4+ T Cell Subset Coexpressing Multiple Markers of HIV Susceptibility.</a> Journal of immunology. , (2011).</li><li>Bere, A., Denny, L., Burgers, W. A., Passmore, 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=Polyclonal+expansion+of+cervical+cytobrush-derived+T+cells+to+investigate+HIV-specific+responses+in+the+female+genital+tract.">Polyclonal expansion of cervical cytobrush-derived T cells to investigate HIV-specific responses in the female genital tract.</a> Immunology. 130, 23-33 (2010).</li><li>Iqbal, S. 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=Elevated+T+cell+counts+and+RANTES+expression+in+the+genital+mucosa+of+HIV-1-resistant+Kenyan+commercial+sex+workers.">Elevated T cell counts and RANTES expression in the genital mucosa of HIV-1-resistant Kenyan commercial sex workers.</a> The Journal of infectious diseases. 192, 728-738 (2005).</li><li>Hirbod, 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=Stable+CD4+expression+and+local+immune+activation+in+the+ectocervical+mucosa+of+HIV-infected+women.">Stable CD4 expression and local immune activation in the ectocervical mucosa of HIV-infected women.</a> Journal of immunology. 191, 3948-3954 (2013).</li><li>Horton, R. E., 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+comparative+analysis+of+gene+expression+patterns+and+cell+phenotypes+between+cervical+and+peripheral+blood+mononuclear+cells.">A comparative analysis of gene expression patterns and cell phenotypes between cervical and peripheral blood mononuclear cells.</a> PloS one. 4, (2009).</li><li>Begaud, E., 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=Reduced+CD4+T+cell+activation+and+in+vitro+susceptibility+to+HIV-1+infection+in+exposed+uninfected+Central+Africans.">Reduced CD4 T cell activation and in vitro susceptibility to HIV-1 infection in exposed uninfected Central Africans.</a> Retrovirology. 3, 35 (2006).</li><li>McLaren, P. J., 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=HIV-exposed+seronegative+commercial+sex+workers+show+a+quiescent+phenotype+in+the+CD4++T+cell+compartment+and+reduced+expression+of+HIV-dependent+host+factors.">HIV-exposed seronegative commercial sex workers show a quiescent phenotype in the CD4+ T cell compartment and reduced expression of HIV-dependent host factors.</a> The Journal of infectious diseases. 202 Suppl 3, (2010).</li><li>Card, C. 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=Reduced+Cellular+Susceptibility+to+In+Vitro+HIV+Infection+Is+Associated+with+CD4T+Cell+Quiescence.">Reduced Cellular Susceptibility to In Vitro HIV Infection Is Associated with CD4T Cell Quiescence.</a> PloS one. 7, (2012).</li><li>Prodger, J. 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=Foreskin+T-cell+subsets+differ+substantially+from+blood+with+respect+to+HIV+co-receptor+expression,+inflammatory+profile,+and+memory+status.">Foreskin T-cell subsets differ substantially from blood with respect to HIV co-receptor expression, inflammatory profile, and memory status.</a> Mucosal immunology. 5, 121-128 (2012).</li><li>Reusch, L. 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=Nonlinear+optical+microscopy+and+ultrasound+imaging+of+human+cervical+structure.">Nonlinear optical microscopy and ultrasound imaging of human cervical structure.</a> Journal of biomedical optics. 18, (2013).</li><li>Oertelt-Prigione, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+and+the+menstrual+cycle.">Immunology and the menstrual cycle.</a> Autoimmunity reviews. 11, (2012).</li><li>Rahman, 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=Mucosal+serpin+A1+and+A3+levels+in+HIV+highly+exposed+sero-negative+women+are+affected+by+the+menstrual+cycle+and+hormonal+contraceptives+but+are+independent+of+epidemiological+confounders.">Mucosal serpin A1 and A3 levels in HIV highly exposed sero-negative women are affected by the menstrual cycle and hormonal contraceptives but are independent of epidemiological confounders.</a> American journal of reproductive immunology. 69, 64-72 (2013).</li></ol>

Given the large gaps in knowledge with respect to immunity at the female genital tract (FGT), phenotypic analysis of CMCs can provide a wide array of insights into multiple lymphocyte populations at the cervix. Coupled with proteomic analysis and viral load measurements in cervical lavage, immunity to sexually transmitted infections (STI)s and other pathogens can be dissected in various populations.
Technical considerations - CMCs: The isolation and successful staining of CMC ...

The majority of new HIV infections worldwide arise through heterosexual transmission, with women representing 47% of new infections in 2011 (UNAIDS1). Understanding the female genital tract (FGT), one of the main entry portals for HIV and other sexually transmitted pathogens, is of high importance on the path to finding efficient strategies to prevent infection. Immune responses at the genital mucosa are clearly unique and differ from those measured in peripheral blood2. However, current knowledge of the immune dynamics at the FGT is limited at best. To date, studies of the mucosal immune environment have largely focused on the gut-associated lymphoid tissues (GALT), where it has become clear that the early events in mucosal tissues following infection have a strong impact on subsequent disease progression3,4. Collecting samples from the genital mucosa represents a great challenge and is at least partially responsible for the lack of understanding of the immunology of the FGT. Solving the puzzle of the immune dynamic between host and pathogen in the context of the distinct environment that is the FGT necessitates efficient methods for collecting and analyzing samples from this locale.
The FGT is divided into two sections: the upper reproductive tract that includes the fallopian tubes, endometrium and endocervix, and the lower tract which contains the ectocervix and the vagina (reviewed by Kaushic et al5). It is still unclear what the relative contribution of these different sites is to HIV infection, but it is believed that both sites could contribute to HIV entry6. T cells represent 40-50% of the leucocytes in the upper and lower reproductive tracts, while macrophages comprise approximately 10% (reviewed in Rodriguez-Garcia et al2). T cells can be detected in the vagina, cervix, and endometrium. Macrophages are more strongly localized in the endometrium and myometrial connective tissue than the cervix, although they can be detected in both tissues. Finally, plasmacytoid dendritic cells (pDCs) and Langerhans cells can also be detected in FGT tissues. The phenotype and proportions of immune populations and their susceptibility to HIV infection may vary importantly according to hormonal cycles, the use of hormonal contraceptives, bacterial vaginosis or sexual activities5,7-9.
Diverse methods have been developed to study the immune populations and environment of the FGT. Cervical biopsy, cervical cytobrushes and cervicovaginal lavages (CVL)10-12 are the most commonly used across the literature. CVL collection by PBS lavage is the simplest method and allows the study of immune modulatory proteins but results in extremely low cell yield, and is therefore not suitable for studying the immune cell populations of the FGT13. CVL samples are, on the other hand, very useful for evaluating the immune environment of the FGT by measuring the expression of various cytokines, chemokines or antimicrobial factors using methods such as ELISA, cytokine bead array14 or mass spectrometry15,16. Characterization of immune cell frequencies, phenotypes and functions can be achieved by collecting cervical mononuclear cells (CMC) by cervical cytobrush or by cervical biopsy sampling.
Cervical biopsy sampling is an invasive method that increases the discomfort and risk of bleeding and takes 2 to 11 days to heal following the procedure depending on the immune status of the woman12. On the other hand, cervical cytobrushes, despite the lower yield of cells collected, is a less invasive and more convenient method to collect immune cells from the FGT. Both methods can reach the same yield of CD45+ leucocytes, but two sequential cervical cytobrushes are necessary to obtain the same amount of cells contained in one biopsy13. Nonetheless, cytobrush sampling still provides an acceptable number of cells (about 5,000 CD45+ cells/cytobrush) for further ex vivo phenotyping by flow cytometry14. Also, functional characterization can be carried out on these samples, as stimulation and intracellular flow cytometry or qPCR have been performed using cytobrush-derived CMCs to identify HIV-specific immune responses17 or Th cell polarization18. Expansion of the T cells population may also facilitate functional studies with CMCs19.
It is important to note that biopsies and cytobrushes sample distinct portions of the FGT. Biopsies are derived from the superior portion of the epithelium and stroma of the ectocervix12,13, while cervical cytobrushes sample the cervical os, collecting cells derived from the epithelium of endocervix and presumably the transformation zone. Cytobrush samples therefore sample a region composed of a single layer of columnar epithelium, while biopsies, include a region lined by a squamous stratified epithelium5. As a result, the nature of the leucocyte populations collected by cervical biopsy and cytobrush differs. Biopsies collect a higher proportion of CD3+ T cells, whereas cytobrushes result in collection of a higher proportion of CD14+ monocytes/macrophages13.
Studying the immunology of the FGT has been an interest for many years20-22 and we have accumulated a great deal of expertise with the study of cytobrush-derived CMCs. Our studies focus mostly on the study of HIV-infected, uninfected and HIV-exposed seronegative (HESN) female sex workers from Nairobi, Kenya. HIV preferentially replicates in activated T cells23 and lower numbers of activated cells that can be targeted by HIV in the FGT could contribute to protection against HIV acquisition. In line with this hypothesis, several studies have described lower immune activation among HESN sex workers who are highly exposed to HIV yet remain uninfected24,25, and this quiescent phenotype is also observed in the FGT14. Here, we describe methodology for processing and assessing T cells activation in CMC samples derived from cervical cytobrushes by ex vivo flow cytometry.

<table border="0" cellpadding="0" cellspacing="0" width="768">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name of Material/ Equipment</td>
			<td style="width:136px;">Company</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:205px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">100uM Cell Strainer for 50 ml Falcon tube</td>
			<td style="width:136px;">BD</td>
			<td style="width:133px;">352360</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RPMI 1640</td>
			<td style="width:136px;">Hyclone</td>
			<td style="width:133px;">SH30027.01</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fetal Bovine serum&#160;</td>
			<td style="width:136px;">Life technology</td>
			<td style="width:133px;">16000044</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fungizone</td>
			<td style="width:136px;">Life technology</td>
			<td style="width:133px;">15290-018</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Penicillin/streptomycin</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:133px;">P4333-20ml</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">50ml Falcon tube</td>
			<td style="width:136px;">Fisher</td>
			<td style="width:133px;">14-959-49A</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Blood Bank disposable transfer pipette</td>
			<td style="width:136px;">Fisher&#160;</td>
			<td style="width:133px;">13-711-6M</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cytobrush plus</td>
			<td style="width:136px;">Cooper surgical</td>
			<td style="width:133px;">C0121</td>
			<td>CMC sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Disposable cervical scraper</td>
			<td style="width:136px;">Quick medical</td>
			<td style="width:133px;">2183</td>
			<td>CMC sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">15 ml Falcon tube&#160;</td>
			<td style="width:136px;">Fisher&#160;</td>
			<td style="width:133px;">14-959-70c</td>
			<td>CVL processsing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">1.5ml tube ependroff</td>
			<td style="width:136px;">Fisher</td>
			<td style="width:133px;">05-402-18</td>
			<td>CVL storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Cell Stain Kit</td>
			<td style="width:136px;">Invitrogen</td>
			<td>Various</td>
			<td>Flow cytometry reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation Buffer (4% PFA)</td>
			<td style="width:136px;">BD</td>
			<td>554655</td>
			<td>Flow cytometry regeant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">IgG mouse&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:133px;">I8765</td>
			<td>Flow cytometry regeant&#160;</td>
		</tr>
	</tbody>
</table>

collection, isolation, and flow cytometric analysis of human endocervical samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

Multiparameter flow cytometry is a powerful tool to dissect the phenotypes and functions of cell subsets in previously uncharacterized tissues. Analysis of CMC samples can yield information on both lymphocyte and monocyte populations with appropriate gating strategies.
A representative CMC gating strategy, compared to a matched PBMC profile, is shown in Figure 2. The FSC-A versus FSC-H plot allows for the exclusion of cell doublets, which are highly prevalent in CMC samples co...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

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Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...