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

Summary

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.

Abstract

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.

Introduction

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 lym....

Protocol

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

1. 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 ºC for several months.
2. Pre-aliquot 5 ml PBS into 50 ml falcon tube (one per sample to be collected) and store at 4 ºC until samp....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

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