Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay

Abstract

Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R = 0.9476, P < 0.0001). Using this assay, we report enumeration of total CD45+ leukocytes, CD4+ and CD8+ T cells, B cells, NK cells, CD14+ monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.

Relationships between IL-17+ Subsets, Tregs and pDCs That Distinguish among SIV Infected Elite Controllers, Low, Medium and High Viral Load Rhesus Macaques

“…A and B). However, in the case of colo-rectal biopsies, unfortunately we could not utilize the technique previously described by Reeves et al [29] to determine absolute numbers because the addition of the beads inhibited the ability to optimally activate the cells in vitro. In addition, the use of weight of the biopsy material as a relative measure was not found to be reliable with a large variation due to differences in cellular and moisture content.…”

Multi‐functional plasmacytoid dendritic cells redistribute to gut tissues during simian immunodeficiency virus infection

“…Macaque peripheral blood mononuclear cells were isolated from EDTA‐treated blood by density gradient centrifugation over lymphocyte separation medium (MP Biomedicals, Solon, OH) and contaminating red blood cells were lysed using a hypotonic ammonium chloride solution. Mononuclear cells were isolated from various tissue sections by both enzymatic and mechanical disruption as described previously for our laboratory …”

“…Mononuclear cells were isolated from various tissue sections by both enzymatic and mechanical disruption as described previously for our laboratory. 5,23,24 Antibodies and flow cytometric analyses Flow cytometry staining of mucosal dendritic cells was performed as previously described. 6 LIVE/DEAD Aqua dye (Invitrogen, Carlsbad, CA) and isotype-matched controls and/or fluorescence-minus-one controls were included for all assays.…”

Obtaining cells from colon of dog with leishmaniasis for flow cytometric analysis

“…Flow cytometry is a powerful tool that can be used to characterize isolated mucosal immune cells phenotypically and to determine the relative abundance of specific cell types. Histological techniques are preferred for determining the anatomical locations of cell populations in situ 1 . Flow cytometry analysis of the cell population of the lamina propria of colonic mucosa provides information pertaining to immune cells that are not easily identifiable in tissue sections.…”