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 for several months.</li>
	<li>Pre-aliquot 5 ml&#160;PBS into 50 ml&#160;falcon tube (one per sample to be collected) and store at 4 &#186;C until sample collection time.</li>
	<li>Label 2-4 cryovials or 1.5 ml&#160;microcentrifuge tubes per sample to collect lavage for storage.</li>
	<li>Prepare cell culture media: RPMI 1640 + 1% penicillin/streptomycin/amphotericin B (final concentration 100 units/ml, 100 &#956;g/ml&#160;and 250 ng/ml, respectively, with no FCS/FBS added.</li>
</ol>
2. Collection of Cytobrush Samples
Collection of cytobrush samples is a non-invasive procedure that must be performed by a trained MD or gynecologist.
<ol>
	<li>Collect cervical mononuclear cells (CMC) using both a cytobrush and a wooden or plastic scraper. Collect CMC from the participant under speculum examination by inserting the scraper and rotating around the cervical os (Figure 1). Insert the cytobrush into the endocervical os, rotate 360&#176;, and immediately place both the cytobrush and scraper into the 50 ml falcon tube containing 5 ml of PBS.</li>
	<li>Keep samples on ice/at 4 &#186;C until processing. To maintain cell viability, process samples within 2 hr&#160;of collection.</li>
	<li>If possible, collect matched blood samples in green top heparin vacutainers for peripheral blood mononuclear cell (PBMC) isolation to serve as a comparator/control for CMC analysis.</li>
</ol>
3. Isolation of CMCs
Perform sample processing in a biosafety level 2 laboratory, in a sterile biosafety cabinet with double gloves.
<ol>
	<li>During collection, CMC samples can become contaminated with blood. Exclude samples with visible blood contamination from the study in order to prevent confounding inclusion of PBMC in the final sample. Due to the low number of lymphocytes isolated from CMC samples, minor blood contamination can easily overwhelm the CMC lymphocyte component.</li>
	<li>Vortex falcon tubes containing both the cytobrush and scraper for 45 sec&#160;to dissociate cells from the brushes. Samples may become foamy during this process; this is normal.</li>
	<li>Use the cytobrush to scrape any remaining material off the scraper, and discard the scraper into a bleach solution. To collect any cells remaining attached to the brush/scraper, use a fresh glove to squeeze the scraper between the thumb and forefinger. Slide down the brush, allowing the cells and PBS to be collected in the same 50 ml tube. Discard the cytobrush into a bleach solution, and discard the glove. 
	IMPORTANT: Use a new glove for each sample.</li>
	<li>Fit a 100 &#956;m&#160;nylon cell strainer to a fresh 50 ml&#160;falcon tube. Using a transfer pipette, collect the PBS-cell suspension from the sample tube and filter through the strainer into the fresh tube.</li>
	<li>Add 5 ml&#160;of RPMI 1640 to the original sample tube. Using the transfer pipette, wash the sides of the sample tube with the RPMI, and transfer through the filter to the new collection tube. Wash the bottom of the nylon filter with the RPMI as well.</li>
	<li>Centrifuge the samples for 10 min at 514 x g. It is crucial to leave the centrifuge brake off during this step, in order to prevent dislodging of the CMC pellet.</li>
	<li>Gently remove the supernatant from the tube, taking care to not disturb the pellet. Pellet size will be highly variable; in some samples, the pellet is quite large due to the presence of large numbers of epithelial cells, while in other cases, the pellet is barely visible and highly transparent.</li>
	<li>Resuspend the pellet by gentle agitation of the falcon tube. Add 5 ml&#160;of PBS to wash the sample.</li>
	<li>Centrifuge again for 10 min at 514 x g with no centrifuge brake.</li>
	<li>Carefully discard the supernatant and re-suspend the pellet as above. The cells are ready for either cryopreservation (using a PBMC cryopreservation protocol), stimulation or flow cytometry phenotyping.</li>
</ol>
4. CMC Surface Staining and Flow Cytometry
<ol>
	<li>Resuspend the pellet from step 3.10 in 100 &#956;l of blocking solution and transfer either into a 96-well plate or FACS tube. The blocking solution (to block Fc&#947;&#160;receptors) recipe is as follows: 1.8 &#956;l&#160;mouse IgG (final concentration 0.2 &#956;g/&#956;l), 5 &#956;l&#160;FBS and 93.2 &#956;l&#160;FACS wash (PBS+2%FBS). Staining can be performed in a 96 well plate if pellet sizes are small enough, but may need to be performed in FACS tubes if pellets are routinely large. It is important to titrate antibodies accordingly.</li>
	<li>Block the samples for 10 min&#160;on ice/at 4 &#176;C.</li>
	<li>Wash the cells with 100 &#956;l of FACS Wash (PBS+2% FBS) in 96-well plates, or 500 &#956;l&#160;in FACS tubes. Centrifuge at 600 x g for 10 min at 4 &#176;C with the brake on low (or off, if a low brake setting is unavailable).</li>
	<li>Remove the supernatant and re-suspend the cell pellet by agitation. Add viability Stain (Live Dead fixable viability dye). Prepare viability dye according to manufacturer&#8217;s instructions. It may need to be titrated for use in each lab/cytometer setup. Incubate for 30 min&#160;in the dark at 4 &#176;C.</li>
	<li>Wash the cells 100&#160;&#956;l/500 &#956;l&#160;of PBS (for 96-well plate/FACS tubes). Centrifuge at 600 x g for 10 min&#160;with low/no brake. Remove supernatant and resuspend cells.</li>
	<li>Incubate the cells with the cocktail of surface marker antibodies, in a total volume of 100 &#956;l. IMPORTANT: Optimize and titrate each flow cytometry panel prior to sample staining. Incubate cells for 30 min&#160;in the dark at 4 &#176;C.</li>
	<li>Repeat step 4.5. if staining in a 96-well plate, transfer cells to a FACS tube and dilute to a final volume of 750 &#181;l of FACS Wash containing 1% paraformaldehyde (PFA) (or CytoFix, diluted 1 in 4). Proceed to data acquisition on a flow cytometer.</li>
</ol>
5. Collection and Preparation of Cervical Vaginal Lavage (CVL)
<ol>
	<li>Prior to cytobrush sampling, use a syringe to wash the endocervix with 2 ml of sterile PBS and aspirate the lavage from the posterior fornix.</li>
	<li>Collect the aspirated lavage sample into a 15 ml falcon tube and keep on ice/at 4 &#186;C until processing.</li>
	<li>Centrifuge sample at 400 x g for 7 min&#160;to remove cellular debris.</li>
	<li>Collect the supernatant, aliquot the sample into 1 ml&#160;or 500 &#956;l&#160;aliquots, and store at -70 &#176;C. Aliquots can be thawed for ELISA or bead array analysis of CVL proteins, but avoid multiple freeze/thaw cycles.</li>
	<li>If desired, resuspend the pellet of cellular debris in RNA Later to measure RNA expression by following the manufacturer&#8217;s protocol.</li>
</ol>
6. Data Acquisition
<ol>
	<li>Prepare a set of compensation tubes that matches the antibody panel. Run the compensation tubes to adjust spectral overlap. Run the samples on any multicolor flow cytometer that is configured for the fluorochrome conjugated antibodies used in the panel.</li>
	<li>Acquire a PBMC sample first to adjust Forward Scatter (FSC) and Side Scatter (SSC) voltage in order to detect the lymphocyte population. Try to keep them at 100 on each axis on a linear scale. Running a PBMC control will make it significantly easier to find the CMC lymphocyte population.</li>
	<li>The FSC threshold for CMC samples may need to be increased relative to PBMC samples due to smear up the SSC axis at low FSC values.</li>
	<li>Vortex each tube before acquiring. Acquire the entire tube to maximize the number of lymphocyte gate events collected. Monitor the machine carefully to avoid clogging.</li>
	<li>Export the data into a flow cytometric analysis program such as FlowJo.</li>
</ol>
7. Gating Strategy
<ol>
	<li>Select Forward side scatter high (FSC-H)/Forward side scatter area (FSC-A) to determine the singlet population.</li>
	<li>Select Time versus Fluorescent marker (as example FITC) to control for the quality of the acquisition. Change in flow rate may introduce artifacts in the data. Exclude any area that shows discrepancy in flow rate.</li>
	<li>Identify the lymphocyte population on SSC-A/FSC-A plot. Use a PBMC control to facilitate the identification. Note that the CMC lymphocyte population might not be as clear as the PBMC control.</li>
	<li>Gate on SSC-A/viability dye to exclude dead cells from live cells. Dead cells can non-specifically incorporate the antibodies, which will introduce artifacts.</li>
	<li>Gate on the SSC-A/CD3 to identify the T cell population. From this gate, identify the CD4+ and CD8+ populations.</li>
</ol>

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<li>Douek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV+disease+progression%3A+immune+activation%2C+microbes%2C+and+a+leaky+gut%5BTitle%5D)+AND+%22Topics+in+HIV+medicine+%3A+a+publication+of+the+International+AIDS+Society%2C+USA%22%5BJournal%5D)">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/pubmed?term=((Disturbance+of+the+gut-associated+lymphoid+tissue+is+associated+with+disease+progression+in+chronic+HIV+infection%5BTitle%5D)+AND+%22Seminars+in+immunopathology%22%5BJournal%5D)">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/pubmed?term=((HIV+infection+in+the+female+genital+tract%3A+discrete+influence+of+the+local+mucosal+microenvironment%5BTitle%5D)+AND+%22American+journal+of+reproductive+immunology%22%5BJournal%5D)">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/pubmed?term=((HIV+infection+of+the+genital+mucosa+in+women%5BTitle%5D)+AND+%22Current+HIV%2FAIDS+reports%22%5BJournal%5D)">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/pubmed?term=((Seminal+fluid+induces+leukocyte+recruitment+and+cytokine+and+chemokine+mRNA+expression+in+the+human+cervix+after+coitus%5BTitle%5D)+AND+%22Journal+of+immunology%22%5BJournal%5D)">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>
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<li>Liebenberg, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stability+and+transport+of+cervical+cytobrushes+for+isolation+of+mononuclear+cells+from+the+female+genital+tract%5BTitle%5D)+AND+%22Journal+of+immunological+methods%22%5BJournal%5D)">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/pubmed?term=((In+situ+distribution+of+HIV-binding+CCR5+and+C-type+lectin+receptors+in+the+human+endocervical+mucosa%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((Feasibility+and+safety+of+cervical+biopsy+sampling+for+mucosal+immune+studies+in+female+sex+workers+from+Nairobi%2C+Kenya%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((Optimizing+viable+leukocyte+sampling+from+the+female+genital+tract+for+clinical+trials%3A+an+international+multi-site+study%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((A+distinct+cytokine+and+chemokine+profile+at+the+genital+mucosa+is+associated+with+HIV-1+protection+among+HIV-exposed+seronegative+commercial+sex+workers%5BTitle%5D)+AND+%22Mucosal+immunology%22%5BJournal%5D)">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/pubmed?term=((Comprehensive+Proteomic+Study+Identifies+Serpin+and+Cystatin+Antiproteases+as+Novel+Correlates+of+HIV-1+Resistance+in+the+Cervicovaginal+Mucosa+of+Female+Sex+Workers%5BTitle%5D)+AND+%22Journal+of+proteome+research%22%5BJournal%5D)">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/pubmed?term=((A+systems+biology+examination+of+the+human+female+genital+tract+shows+compartmentalization+of+immune+factor+expression%5BTitle%5D)+AND+%22Journal+of+virology%22%5BJournal%5D)">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/pubmed?term=((HIV-specific+T+cell+responses+detected+in+the+genital+tract+of+chronically+HIV-infected+women+are+largely+monofunctional%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+a+Human+Cervical+CD4%2B+T+Cell+Subset+Coexpressing+Multiple+Markers+of+HIV+Susceptibility%5BTitle%5D)+AND+%22Journal+of+immunology%22%5BJournal%5D)">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/pubmed?term=((Polyclonal+expansion+of+cervical+cytobrush-derived+T+cells+to+investigate+HIV-specific+responses+in+the+female+genital+tract%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">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/pubmed?term=((Elevated+T+cell+counts+and+RANTES+expression+in+the+genital+mucosa+of+HIV-1-resistant+Kenyan+commercial+sex+workers%5BTitle%5D)+AND+%22The+Journal+of+infectious+diseases%22%5BJournal%5D)">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/pubmed?term=((Stable+CD4+expression+and+local+immune+activation+in+the+ectocervical+mucosa+of+HIV-infected+women%5BTitle%5D)+AND+%22Journal+of+immunology%22%5BJournal%5D)">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/pubmed?term=((A+comparative+analysis+of+gene+expression+patterns+and+cell+phenotypes+between+cervical+and+peripheral+blood+mononuclear+cells%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((Reduced+CD4+T+cell+activation+and+in+vitro+susceptibility+to+HIV-1+infection+in+exposed+uninfected+Central+Africans%5BTitle%5D)+AND+%22Retrovirology%22%5BJournal%5D)">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/pubmed?term=((HIV-exposed+seronegative+commercial+sex+workers+show+a+quiescent+phenotype+in+the+CD4%2B+T+cell+compartment+and+reduced+expression+of+HIV-dependent+host+factors%5BTitle%5D)+AND+%22The+Journal+of+infectious+diseases%22%5BJournal%5D)">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/pubmed?term=((Reduced+Cellular+Susceptibility+to+In+Vitro+HIV+Infection+Is+Associated+with+CD4T+Cell+Quiescence%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((Foreskin+T-cell+subsets+differ+substantially+from+blood+with+respect+to+HIV+co-receptor+expression%2C+inflammatory+profile%2C+and+memory+status%5BTitle%5D)+AND+%22Mucosal+immunology%22%5BJournal%5D)">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/pubmed?term=((Nonlinear+optical+microscopy+and+ultrasound+imaging+of+human+cervical+structure%5BTitle%5D)+AND+%22Journal+of+biomedical+optics%22%5BJournal%5D)">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/pubmed?term=((Immunology+and+the+menstrual+cycle%5BTitle%5D)+AND+%22Autoimmunity+reviews%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22American+journal+of+reproductive+immunology%22%5BJournal%5D)">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>

The authors have nothing to disclose.

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><tbody><tr><td>100 &amp;mu;m cell strainer for 50 ml Falcon tube</td><td>BD</td><td>352360</td><td>CMC processing</td></tr><tr><td>RPMI 1640</td><td>Hyclone</td><td>SH30027.01</td><td>CMC processing</td></tr><tr><td>Fetal bovine serum</td><td>Life technology</td><td>16000044</td><td>CMC processing</td></tr><tr><td>Fungizone</td><td>Life technology</td><td>15290-018</td><td>CMC processing</td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma</td><td>P4333-20ml</td><td>CMC processing</td></tr><tr><td>50 ml Falcon tube</td><td>Fisher</td><td>14-959-49A</td><td>CMC processing</td></tr><tr><td>Blood Bank disposable transfer pipette</td><td>Fisher</td><td>13-711-6M</td><td>CMC processing</td></tr><tr><td>Cytobrush plus</td><td>Cooper surgical</td><td>C0121</td><td>CMC sampling</td></tr><tr><td>Disposable cervical scraper</td><td>Quick medical</td><td>2183</td><td>CMC sampling</td></tr><tr><td>15 ml Falcon tube</td><td>Fisher</td><td>14-959-70c</td><td>CVL processsing</td></tr><tr><td>1.5 ml tube Eppendorf</td><td>Fisher</td><td>05-402-18</td><td>CVL storage</td></tr><tr><td>LIVE/DEAD Fixable Cell Stain Kit</td><td>Invitrogen</td><td>Various</td><td>Flow cytometry reagent</td></tr><tr><td>Fixation buffer (4% PFA)</td><td>BD</td><td>554655</td><td>Flow cytometry regeant</td></tr><tr><td>IgG mouse</td><td>Sigma</td><td>I8765</td><td>Flow cytometry regeant</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...

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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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Research

JoVE Journal

Medicine

19.1K Views.  University of Manitoba. 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. 

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

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Cancer Research

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Immunology and Infection

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if fertilization is successful, the embryo. The epithelial cells that line the inner surface of fallopian tubes are integral to both normal and abnormal fallopian tube processes, including disease initiation. After menopause, fallopian tubes cease to have a significant role in the body and the intraepithelial cell makeup changes. We describe a method in which these epithelial cells can be isolated from fresh fallopian tubes with minimal stromal cell contamination into a single-cell suspension. These cells can be grown in culture or used for further analysis such as flow cytometry and single-cell RNA sequencing. This isolation protocol can be achieved in 4-6 h and yields viable cells that can be used for immediate downstream analysis. This efficient protocol facilitates the isolation of fallopian tube epithelial cells with an enriched epithelial population.

This protocol describes a one-day method for the isolation of human fallopian tube epithelial cells. Isolated epithelial cells can be plated in 2-dimensional (2D) culture or dissociated into single-cell suspensions and utilized in downstream experiments, including flow cytometry and single-cell RNA sequencing.

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility.
With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 &#176;C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5&#8242;,6,6&#8242;-tetrachloro-1,1&#8242;,3,3&#8242;-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no ...

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

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