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AIDS Res Hum Retroviruses. 2017 Nov;33(S1):S31-S39. doi: 10.1089/AID.2017.0208.

An Optimized and Validated Method for Isolation and Characterization of Lymphocytes from HIV+ Human Gut Biopsies.

Author information

1
1 Gladstone Institute of Virology and Immunology, Gladstone Institutes , San Francisco, California.
2
2 Department of Urology, University of California , San Francisco, San Francisco, California.
3
3 San Francisco VA Health Care System and University of California , San Francisco (UCSF), San Francisco, California.
4
4 Positive Health Program, Department of Medicine, University of California , San Francisco, San Francisco, California.
5
5 Division of HIV, Infectious Diseases, and Global Medicine, Department of Medicine, University of California , San Francisco, San Francisco, California.
6
6 Division of Gastroenterology, Department of Medicine, University of California , San Francisco, San Francisco, California.
7
7 Division of Experimental Medicine, University of California , San Francisco, San Francisco, California.
8
8 Department of Microbiology and Immunology, University of California , San Francisco, San Francisco, California.

Abstract

The gastrointestinal (GI) tract harbors most of the body's immune cells and is also a major HIV reservoir in ART-treated patients. To achieve a cure, most HIV-infected cells must be identified and eliminated. While obtaining gut biopsies is a relatively noninvasive method of sampling relevant tissue for monitoring HIV activity, immune cell isolation from these limited tissue samples has proven to be challenging. Enzymatic tissue digestion is required for maximal immune cell isolation from gut biopsies. However, these enzymatic digestions can also be detrimental for preservation of cellular surface markers that are required for accurate identification of various subsets of leukocytes. In this study, we describe an optimized protocol for isolation of lymphocytes from human gut biopsies. We also discuss our validation results, which show that compared with several other collagenase preparations, the use of CSLPA maintains high lymphocyte recovery while preserving the integrity of most cellular surface antigens that we tested. Importantly, chemokine receptors that are used to characterize various subsets of T cells, which are notorious for being digested during a typical enzymatic tissue digestion, are highly preserved using this protocol.

KEYWORDS:

CXCR5; CyTOF; collagenase; human gut biopsies; lymphocyte isolation; surface antigens

PMID:
28882052
PMCID:
PMC5684666
DOI:
10.1089/AID.2017.0208
[Indexed for MEDLINE]
Free PMC Article

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