Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Abstract

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded > 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

IL‐10 expression in macrophages from neonates born from obese mothers is suppressed by IL‐4 and LPS/INFγ

“…Based on the timing necessary to develop a proper isolation protocol all the samples were collected from deliveries that took place in the morning (based on the scheduled time of delivery). Peripheral blood mononuclear cells (PBMC) were separated shortly after birth by Ficoll density gradient (Figueroa et al, ) using Histopaque‐1077 (Sigma–Aldrich, MO) centrifugation and incubated in RPMI 1640 medium (Sigma–Aldrich, MO) and seeded at density of 8–10 × 10 6 cells/ml in 100 mm culture dishes at 5% CO 2 for 2 hr at 37°C. Non‐adherent cells were removed by Phosphate‐buffered saline (PBS) wash. Monocyte identity was assessed by flow cytometry showing that 78% of the cells were CD14 + .…”

Development of Multifunctional Biopolymeric Auto-Fluorescent Micro- and Nanogels as a Platform for Biomedical Applications

“…The washing of the cells with PBS was again done and the total cell number and cell viability were evaluated by trypan blue exclusion (Sigma-Aldrich, St. Louis, MO, United States) in a hemocytometer counting chamber. Finally, the cells were re-suspended in complete culture medium containing Roswell Park Memorial Institute (RPMI) 1640 (Life Technologies, Gaithersburg, MD, United States), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, St. Louis, MO, United States), 2 mM glutamine (Sigma-Aldrich, St. Louis, MO, United States), 100 µg streptomycin (Sigma-Aldrich, St. Louis, MO, United States), 100 U penicillin (Sigma-Aldrich, St. Louis, MO, United States), and 10% fetal bovine serum (Life Technologies, Gaithersburg, MD, United States) (Figueroa et al, 2016).…”

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

“…CD14-positive monocytes are abundant in peripheral blood, representing about 10% of circulating leukocytes (Auffray et al, 2009), and can be differentiated ex vivo into monocyte-derived macrophages (MDMs) or monocyte-derived dendritic cells (MoDCs) (Figueroa et al, 2016;Jin and Kruth, 2016), making them the ideal starting point for generation of isogenic primary myeloid cells. Monocytes are isolated from donor blood and immediately subjected to CRISPR-Cas9 ribonucleoprotein (RNP) nucleofection.…”