An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

Published: March 29, 2024

Abstract

Source: Hsu, P., et al. Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs). J. Vis. Exp. (2015)

This video demonstrates an in vitro assay to explore the immunomodulatory influence of mesenchymal stem cells (MSCs) on effector T cell proliferation. The assay includes co-culturing MSCs with PBMCs to induce the monocyte population to acquire an immunomodulatory phenotype, leading to the secretion of interleukin-10 (IL-10). Following incubation with activated CD4+ T cells, the induced monocytes effectively suppress T cell proliferation.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Density Gradient Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) Add 25 ml heparinized whole blood into a 50 ml tube by a 25 ml pipette. Dilute cells with 25 ml of phosphate-buffered saline (PBS). Add 15 ml of Ficoll-Paque density gradient into a new 50 ml tube, and while tilting …

Divulgaciones

The authors have nothing to disclose.

Materials

Ficoll-Paque PLUS GE Healthcare 71-7167-00 AG Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)
Vibrant CFDA-SE Cell Tracer Kit (CFSE) Life Technologies V12883 Cellular label for detection of cell division
Dynabeads Human T-Activator CD3/28 Life Technologies 111.32D Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc.
autoMACS™ Separator Miltenyi Biotec autoMACS™ Separator Magnetic based cell separator
autoMACS® Columns Miltenyi Biotec 130-021-101 separation columns
CD14 microbeads, human  Miltenyi Biotec 130-050-201 For positive selection of CD14+ human monocytes and macrophages from PBMCs
CD4 microbeads, human  Miltenyi Biotec 130-045-101 For positive selection of CD4+ human T lymphocytes from PBMCs
RPMI 1640 Medium Life Technologies 11875 Human PBMC/leukocyte culture medium
DMEM, Low glucose, pyruvate Life Technologies 11885 Human mesenchymal stem cell (MSC) culture medium
L-glutamine Life Technologies 25030-081 Supplementation for MSC complete medium
Penicillin/Streptomycin Life Technologies 15070-063 Supplementation for PBMC/leukocyte and MSC complete medium
Fetal bovine serum (FBS) 1) Hyclone, for MSC culture, 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations) 1) SH30070.03M 2) 10091-148 Pre-test lots for support of MSC in vitro culture
24-well cell culture plate Corning COR3524 Co-culture plate
50 mL centrifuge tube Corning 430291 Isolation PBMCs from whole blood by Ficoll-Paque PLUS
15 mL centrifuge tube  Corning 430766 Collection of the labeled and unlabeled cell fractions
Round-bottom tubes BD Falcon  352008 Collection of cells for flow cytometric analysis

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An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation. J. Vis. Exp. (Pending Publication), e22015, doi: (2024).

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